Oligonucleotide probes and uses thereof

ABSTRACT

Methods and compositions are provided to identify oligonucleotides that bind targets of interest. The targets include tissues, cells, circulating biomarkers such as microvesicles, including those derived from various diseases. The oligonucleotides can be used in diagnostic and therapeutic applications.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.16/084,504, filed Sep. 12, 2018, which is the national stage entry ofInternational Patent Application No. PCT/US2017/023108, filed Mar. 18,2017, which claims the benefit of U.S. Provisional Patent ApplicationNos. 62/310,665, filed Mar. 18, 2016; 62/413,361, filed Oct. 26, 2016;62/420,497, filed Nov. 10, 2016; 62/432,561, filed Dec. 9, 2016;62/457,691, filed Feb. 10, 2017; and 62/472,953, filed Mar. 17, 2017;all of which applications are incorporated herein by reference in theirentirety.

SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The entire content of the following electronic submission of thesequence listing via the USPTO EFS-WEB server, as authorized and setforth in MPEP § 1730 II.B.2(a), is incorporated herein by reference inits entirety for all purposes. The sequence listing is within theelectronically filed text file that is identified as follows:

File Name: SequenceListing.txt

Date of Creation: Jun. 10, 2020

Size (bytes): 39,314,000 bytes

BACKGROUND OF THE INVENTION

The invention relates generally to oligonucleotide probes, which areuseful for diagnostics of cancer and/or other diseases or disorders andas therapeutics to treat such medical conditions. The invention furtherrelates to materials and methods for the administration ofoligonucleotide probes capable of binding to cells of interest.

Oligonucleotide probes, or aptamers, are oligomeric nucleic acidmolecules having specific binding affinity to molecules, which may bethrough interactions other than classic Watson-Crick base pairing.Unless otherwise specified, an “aptamer” as the term is used herein canrefer to nucleic acid molecules that can associate with targets,regardless of manner of target recognition. Unless other specified, theterms “aptamer,” “oligonucleotide,” “polynucleotide,” “oligonucleotideprobe,” or the like may be used interchangeably herein.

Oligonucleotide probes, like peptides generated by phage display ormonoclonal antibodies (“mAbs”), are capable of specifically binding toselected targets and modulating the target's activity, e.g., throughbinding aptamers may block their target's ability to function. Createdby an in vitro selection process from pools of random sequenceoligonucleotides, aptamers have been generated for numerous proteinsincluding growth factors, transcription factors, enzymes,immunoglobulins, and receptors. A typical aptamer is 10-15 kDa in size(30-45 nucleotides), binds its target with sub-nanomolar affinity, anddiscriminates against closely related targets (e.g., aptamers can bedesigned to not bind other proteins from the same gene family). A seriesof structural studies have shown that aptamers are capable of using thesame types of binding interactions (e.g., hydrogen bonding,electrostatic complementarity, hydrophobic contacts, steric exclusion)that drive affinity and specificity in antibody-antigen complexes.

We have previously identified oligonucleotides and libraries ofoligonucleotides useful for the detection of microvesicles in bodilyfluid samples. Microvesicles can be shed by diseased cells, such ascancer cells, into various bodily fluids such as blood. Thus provide ameans of liquid biopsy, including without limitation blood baseddiagnostics. In some cases, tissue samples are available. The presentinvention provides methods of enriching oligonucleotide librariesagainst tissues of interest. Applications of the invention includewithout limitation theranostics (e.g., predicting a drug response) anddiagnostics (e.g., detecting cancer samples). As the methods of theinvention provide aptamers that specifically recognize diseased cells,the aptamers themselves can be used in therapeutic applications.

INCORPORATION BY REFERENCE

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

SUMMARY OF THE INVENTION

Compositions and methods of the invention provide oligonucleotide probesthat recognize tissues having phenotypes of interest. In variousembodiments, oligonucleotide probes of the invention are used indiagnostic, prognostic or theranostic processes to characterize aphenotype of that sample. The diagnosis may be related to a cancer. Inother embodiments, oligonucleotide probes of the invention arechemically modified or composed in a pharmaceutical composition fortherapeutic applications.

In an aspect, the invention provides an oligonucleotide comprising aregion corresponding to: a) a variable sequence as described in any oneof Examples 19-31; b) a variable sequence as described in any one ofTables 20-23, 25, 27, 38-40, or 45; or c) a sequence listed in any oneof SEQ ID NO. 1-206506. In some embodiments, the oligonucleotide furthercomprises a 5′ region with sequence 5′-CTAGCATGACTGCAGTACGT (SEQ ID NO.4), a 3′ region with sequence 5′-CTGTCTCTTATACACATCTGACGCTGCCGACGA (SEQID NO. 5), or both. The invention further provides an oligonucleotidecomprising a nucleic acid sequence or a portion thereof that is at least50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100 percenthomologous to such oligonucleotide sequences. In a related aspect, theinvention provides a plurality of oligonucleotides comprising at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150,175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000,70000, 80000, 90000, or at least 100000 different oligonucleotidesequences as described above.

In an embodiment, the oligonucleotide or the plurality ofoligonucleotides comprise a DNA, RNA, 2′-O-methyl or phosphorothioatebackbone, or any combination thereof. In some embodiments, theoligonucleotide or the plurality of oligonucleotides comprises at leastone of DNA, RNA, PNA, LNA, UNA, and any combination thereof. Theoligonucleotide or at least one member of the plurality ofoligonucleotides can have at least one functional modification selectedfrom the group consisting of DNA, RNA, biotinylation, a non-naturallyoccurring nucleotide, a deletion, an insertion, an addition, and achemical modification. In some embodiments, the chemical modificationcomprises at least one of C18, polyethylene glycol (PEG), PEG4, PEG6,PEG8, PEG12 and digoxygenin.

The oligonucleotide or plurality of oligonucleotides of the inventioncan be labeled. For example, the oligonucleotide or plurality ofoligonucleotides can be attached to a nanoparticle, liposome, gold,magnetic label, fluorescent label, light emitting particle, biotinmoiety, or radioactive label.

In an aspect, the invention provides a method of enriching anoligonucleotide library using multiple rounds of positive and negativeselection. The method of enriching a plurality of oligonucleotides maycomprise: a) performing at least one round of positive selection,wherein the positive selection comprises: i) contacting at least onesample with the plurality of oligonucleotides, wherein the at least onesample comprises tissue; and ii) recovering members of the plurality ofoligonucleotides that associated with the at least one sample; b)optionally performing at least one round of negative selection, whereinthe negative selection comprises: i) contacting at least one additionalsample with the plurality of oligonucleotides, wherein at least oneadditional sample comprises tissue; ii) recovering members of theplurality of oligonucleotides that did not associate with the at leastone additional sample; and c) amplifying the members of the plurality ofoligonucleotides recovered in at least one or step (a)(ii) and step(b)(ii), thereby enriching the oligonucleotide library. In anembodiments, the recovered members of the plurality of oligonucleotidesin step (a)(ii) are used as the input for the next iteration of step(a)(i). In an embodiment, the recovered members of the plurality ofoligonucleotides in step (b)(ii) are used as the input for the nextiteration of step (a)(i). The unenriched oligonucleotide library maypossess great diversity. For example, the unenriched oligonucleotidelibrary may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000,400000, 500000, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵,10¹⁶, 10¹⁷, or at least 10¹⁸ different oligonucleotide sequences. In anembodiment, the unenriched oligonucleotide library comprises the naïveF-Trin library as described herein.

In some embodiments of the enrichment methods of the invention, the atleast one sample and/or at least one additional sample comprise fixedtissue. The fixed tissue may comprise formalin fixed paraffin embedded(FFPE) tissue. In embodiment, the FFPE tissue comprises at least one ofa fixed tissue, unstained slide, bone marrow core or clot, biopsysample, surgical sample, core needle biopsy, malignant fluid, and fineneedle aspirate (FNA). The FFPE tissue can be fixed on a substrate. Forexample, the substrate can be a glass slide or membrane.

In some embodiment, the at least one sample and/or the at least oneadditional sample are fixed on different substrates. Alternately, the atleast one sample and/or the at least one additional sample is fixed on asingle substrate. In some embodiments, the at least one sample and/orthe at least one additional sample are lysed, scraped from a substrate,or subjected to microdissection. Lysed samples can be arrayed on asubstrate. In some embodiments, the substrate comprises a membrane. Forexample, the membrane can be a nitrocellulose membrane.

In various embodiments of the enrichment methods of the invention, theat least one sample and the at least one additional sample differ in aphenotype of interest. The at least one sample and the at least oneadditional sample can be from different sections of a same substrate. Asa non-limiting example, the samples may comprise cancer tissue andnormal adjacent tissue from a fixed tissue sample. In such cases, the atleast one sample and the at least one additional sample may be scrapedor microdissected from the same substrate to facilitate enrichment.

The oligonucleotide library can be enriched for analysis of any desiredphenotype. In embodiments, the phenotype comprises a tissue, anatomicalorigin, medical condition, disease, disorder, or any combinationthereof. For example, the tissue can be muscle, epithelial, connectiveand nervous tissue, or any combination thereof. For example, theanatomical origin can be the stomach, liver, small intestine, largeintestine, rectum, anus, lungs, nose, bronchi, kidneys, urinary bladder,urethra, pituitary gland, pineal gland, adrenal gland, thyroid,pancreas, parathyroid, prostate, heart, blood vessels, lymph node, bonemarrow, thymus, spleen, skin, tongue, nose, eyes, ears, teeth, uterus,vagina, testis, penis, ovaries, breast, mammary glands, brain, spinalcord, nerve, bone, ligament, tendon, or any combination thereof.

In various embodiments of the enrichment methods of the invention, themethod further comprises determining a target of the enriched members ofthe oligonucleotide library. Techniques for such determining areprovided herein.

In an aspect, the invention provides a method of characterizing aphenotype in a sample comprising: a) contacting the sample with at leastone oligonucleotide or plurality of oligonucleotides; and b) identifyinga presence or level of a complex formed between the at least oneoligonucleotide or plurality of oligonucleotides and the sample, whereinthe presence or level is used to characterize the phenotype. In arelated aspect, the invention provides a method of visualizing a samplecomprising: a) contacting the sample with at least one oligonucleotideor plurality of oligonucleotides; b) removing the at least oneoligonucleotide or members of the plurality of oligonucleotides that donot bind the sample; and c) visualizing the at least one oligonucleotideor plurality of oligonucleotides that bound to the sample. Thevisualization can be used to characterize a phenotype.

The sample to be characterized can be any useful sample, includingwithout limitation a tissue sample, bodily fluid, cell, cell culture,microvesicle, or any combination thereof. In some embodiments, thetissue sample comprises fixed tissue. The fixed tissue can be, e.g.,formalin fixed paraffin embedded (FFPE) tissue. In various embodiments,the FFPE sample comprises at least one of a fixed tissue, unstainedslide, bone marrow core or clot, biopsy sample, surgical sample, coreneedle biopsy, malignant fluid, and fine needle aspirate (FNA).

Identifying a presence or level may comprise any useful technique,including without limitation nucleic acid sequencing, amplification,hybridization, gel electrophoresis, chromatography, or visualization. Insome embodiments, the identifying by hybridization comprises contactingthe sample with at least one labeled probe that is configured tohybridize with at least one oligonucleotide or plurality ofoligonucleotides. The at least one labeled probe can be directly orindirectly attached to a label. The label can be, e.g., a fluorescent,radioactive or magnetic label. An indirect label can be, e.g., biotin.In some embodiments, the sequencing comprises next generationsequencing, dye termination sequencing, and/or pyrosequencing of the atleast one oligonucleotide or plurality of oligonucleotides. Thevisualization may be that of a signal linked directly or indirectly tothe at least one oligonucleotide or plurality of oligonucleotides. Thesignal can be any useful signal, e.g., a fluorescent signal or anenzymatic signal. In some embodiments, the enzymatic signal is producedby at least one of a luciferase, firefly luciferase, bacterialluciferase, luciferin, malate dehydrogenase, urease, peroxidase,horseradish peroxidase (HRP), alkaline phosphatase (AP),β-galactosidase, glucoamylase, lysozyme, a saccharide oxidase, glucoseoxidase, galactose oxidase, glucose-6-phosphate dehydrogenase, aheterocyclic oxidase, uricase, xanthine oxidase, lactoperoxidase, andmicroperoxidase. Visualization may comprise use of light microscopy orfluorescent microscopy.

In the methods of the invention directed to characterizing orvisualizing a sample, the target of at least one of the at least oneoligonucleotide or plurality of oligonucleotides may be known. Forexample, an oligonucleotide may bind a known protein target. In someembodiments, the target of at least one the at least one oligonucleotideor plurality of oligonucleotides is unknown. For example, the at leastone oligonucleotide or plurality of oligonucleotides may themselvesprovide a biosignature or other useful result that does not necessarilyrequire knowledge of the antigens bound by some or all of theoligonucleotides.

In the methods of characterizing or visualizing a sample, the at leastone oligonucleotide or plurality of oligonucleotides can be as providedabove. The at least one oligonucleotide or plurality of oligonucleotidesmay have been determined using the enrichment methods of the inventionprovided herein.

For example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids may have a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or allof SEQ ID NOs. 2922-2926, 2929-2947 and 2950-2952. In such cases, thephenotype may be, e.g., lung cancer or prostate cancer.

In another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or allof SEQ ID NOs. 2953-2961 and 2971-2979. In such cases, the phenotype maybe, e.g., prostate cancer.

In yet another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50 or all of SEQ ID NOs. 3039-3061. In such cases, the phenotypemay be, e.g., HER2 status (+/−).

In still another example, the at least one oligonucleotide or pluralityof oligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 150,000 or allof SEQ ID NOs. 3062-103061 and 103062-203061. In such cases, thephenotype may be, e.g., response to anti-HER2 therapy, whereinoptionally the anti-HER2 therapy comprises trastuzumab.

In an example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000 or all of SEQ ID NOs. 203064-203067 and203076-206478. In such cases, the phenotype may be, e.g., response to atleast one of FOLFOX and bevacizumab.

In another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or all ofSEQ ID NOs. 206491-206506. In such cases, the phenotype may be, e.g., atissue identity, including without limitation whether the tissuecomprises breast, colon, kidney, lung or pancreatic tissue.

In the methods of the invention, including enriching an oligonucleotidelibrary, characterizing a sample or visualizing a sample, the phenotypecan be a biomarker status. In some embodiments, the biomarker isselected from Table 4. In some embodiments, the biomarker statuscomprises at least one of HER2 positive, HER2 negative, EGFR positive,EGFR negative, TUBB3 positive, or TUBB3 negative. In some embodiments,the biomarker status comprises expression, copy number, mutation,insertion, deletion or other alteration of at least one of ALK, AR, ER,ERCC1, Her2/Neu, MGMT, MLH1, MSH2, MSH6, PD-1, PD-L1, PD-L1 (22c3),PMS2, PR, PTEN, RRM1, TLE3, TOP2A, TOPO1, TrkA, TrkB, TrkC, TS, andTUBB3. In various embodiments, the biomarker status comprises thepresence or absence of at least one of EGFR vIII or MET Exon 14Skipping. In embodiments, the biomarker status comprises expression,copy number, fusion, mutation, insertion, deletion or other alterationof at least one of ALK, BRAF, NTRK1, NTRK2, NTRK3, RET, ROS1, and RSPO3.In embodiments, the biomarker status comprises expression, copy number,fusion, mutation, insertion, deletion or other alteration of at leastone of ABL2, ACSL3, ACSL6, AFF1, AFF3, AFF4, AKAP9, AKT2, AKT3, ALDH2,ALK, APC, ARFRP1, ARHGAP26, ARHGEF12, ARID1A, ARID2, ARNT, ASPSCR1,ASXL1, ATF1, ATIC, ATM, ATP1A1, ATR, AURKA, AURKB, AXIN1, AXL, BAP1,BARD1, BCL10, BCL11A, BCL2L11, BCL3, BCL6, BCL7A, BCL9, BCR, BIRC3, BLM,BMPR1A, BRAF, BRCA1, BRCA2, BRIP1, BUB1B, C11orf30 (EMSY), C2orf44,CACNA1D, CALR, CAMTA1, CANT1, CARD11, CARS, CASC5, CASP8, CBFA2T3, CBFB,CBL, CBLB, CCDC6, CCNB11P1, CCND1, CCND2, CCND3, CCNE1, CD274 (PDL1),CD74, CD79A, CDC73, CDH11, CDK4, CDK6, CDK8, CDKN1B, CDKN2A, CDX2,CHEK1, CHEK2, CHIC2, CHN1, CIC, CIITA, CLP1, CLTC, CLTCL1, CNBP, CNTRL,COPB1, CREB1, CREB3L1, CREB3L2, CREBBP, CRKL, CRTC1, CRTC3, CSF1R,CSF3R, CTCF, CTLA4, CTNNA1, CTNNB1, CYLD, CYP2D6, DAXX, DDR2, DDX10,DDX5, DDX6, DEK, DICER1, DOT1L, EBF1, ECT2L, EGFR, ELK4, ELL, EML4,EP300, EPHA3, EPHA5, EPHB1, EPS15, ERBB2 (HER2), ERBB3 (HER3), ERBB4(HER4), ERC1, ERCC2, ERCC3, ERCC4, ERCC5, ERG, ESR1, ETV1, ETV5, ETV6,EWSR1, EXT1, EXT2, EZH2, EZR, FANCA, FANCC, FANCD2, FANCE, FANCG, FANCL,FAS, FBXO11, FBXW7, FCRL4, FGF10, FGF14, FGF19, FGF23, FGF3, FGF4, FGF6,FGFR1, FGFR1OP, FGFR2, FGFR3, FGFR4, FH, FHIT, FIP1L1, FLCN, FLI1, FLT1,FLT3, FLT4, FNBP1, FOXA1, FOXO1, FOXP1, FUBP1, FUS, GAS7, GATA3, GID4(C17orf39), GMPS, GNA13, GNAQ, GNAS, GOLGA5, GOPC, GPHN, GPR124, GRIN2A,GSK3B, H3F3A, H3F3B, HERPUD1, HGF, HIP1, HMGA1, HMGA2, HNRNPA2B1, HOOK3,HSP90AA1, HSP90AB1, IDH1, IDH2, IGF1R, IKZF1, IL2, IL21R, IL6ST, IL7R,IRF4, ITK, JAK1, JAK2, JAK3, JAZF1, KDM5A, KDR (VEGFR2), KEAP1,KIAA1549, KIF5B, KIT, KLHL6, KMT2A (MLL), KMT2C (MLL3), KMT2D (MLL2),KRAS, KTN1, LCK, LCP1, LGR5, LHFP, LIFR, LPP, LRIG3, LRP1B, LYL1, MAF,MALT1, MAML2, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MCL1, MDM2, MDM4, MDS2,MEF2B, MEN1, MET (cMET), MITF, MLF1, MLH1 (NGS), MLLT1, MLLT10, MLLT3,MLLT4, MLLT6, MNX1, MRE11A, MSH2 (NGS), MSH6 (NGS), MSI2, MTOR, MYB,MYC, MYCN, MYD88, MYH11, MYH9, NACA, NCKIPSD, NCOA1, NCOA2, NCOA4, NF1,NF2, NFE2L2, NFIB, NFKB2, NFKBIA, NIN, NOTCH2, NPM1, NR4A3, NSD1, NT5C2,NTRK1, NTRK2, NTRK3, NUP214, NUP93, NUP98, NUTM1, PALB2, PAX3, PAX5,PAX7, PBRM1, PBX1, PCM1, PCSK7, PDCD1 (PD1), PDCD1LG2 (PDL2), PDGFB,PDGFRA, PDGFRB, PDK1, PER1, PICALM, PIK3CA, PIK3R1, PIK3R2, PIM1, PML,PMS2 (NGS), POLE, POT1, POU2AF1, PPARG, PRCC, PRDM1, PRDM16, PRKAR1A,PRRX1, PSIP1, PTCH1, PTEN (NGS), PTPN11, PTPRC, RABEP1, RAC1, RAD50,RAD51, RAD51B, RAF1, RALGDS, RANBP17, RAP1GDS1, RARA, RB1, RBM15, REL,RET, RICTOR, RMI2, RNF43, ROS1, RPL22, RPL5, RPN1, RPTOR, RUNX1,RUNX1T1, SBDS, SDC4, SDHAF2, SDHB, SDHC, SDHD, SEPT9, SET, SETBP1,SETD2, SF3B1, SH2B3, SH3GL1, SLC34A2, SMAD2, SMAD4, SMARCB1, SMARCE1,SMO, SNX29, SOX10, SPECC1, SPEN, SRGAP3, SRSF2, SRSF3, SS18, SS18L1,STAT3, STAT4, STAT5B, STIL, STK11, SUFU, SUZ12, SYK, TAF15, TCF12, TCF3,TCF7L2, TET1, TET2, TFEB, TFG, TFRC, TGFBR2, TLX1, TNFAIP3, TNFRSF14,TNFRSF17, TOP1, TP53, TPM3, TPM4, TPR, TRAF7, TRIM26, TRIM27, TRIM33,TRIP11, TRRAP, TSC1, TSC2, TSHR, TTL, U2AF1, USP6, VEGFA, VEGFB, VTI1A,WHSC1, WHSC1L1, WIF1, WISP3, WRN, WT1, WWTR1, XPA, XPC, XPO1, YWHAE,ZMYM2, ZNF217, ZNF331, ZNF384, ZNF521, and ZNF703. The biomarker statusmay comprise expression, copy number, fusion, mutation, insertion,deletion or other alteration of at least one of ABI1, ABL1, ACKR3, AKT1,AMER1 (FAM123B), AR, ARAF, ATP2B3, ATRX, BCL11B, BCL2, BCL2L2, BCOR,BCORL1, BRD3, BRD4, BTG1, BTK, C15orf65, CBLC, CD79B, CDH1, CDK12,CDKN2B, CDKN2C, CEBPA, CHCHD7, CNOT3, COL1A1, COX6C, CRLF2, DDB2, DDIT3,DNM2, DNMT3A, EIF4A2, ELF4, ELN, ERCC1 (NGS), ETV4, FAM46C, FANCF, FEV,FOXL2, FOXO3, FOXO4, FSTL3, GATA1, GATA2, GNA11, GPC3, HEY1, HIST1H3B,HIST1H4I, HLF, HMGN2P46, HNF1A, HOXA11, HOXA13, HOXA9, HOXC11, HOXC13,HOXD11, HOXD13, HRAS, IKBKE, INHBA, IRS2, JUN, KAT6A (MYST3), KAT6B,KCNJ5, KDM5C, KDM6A, KDSR, KLF4, KLK2, LASP1, LMO1, LMO2, MAFB, MAX,MECOM, MED12, MKL1, MLLT11, MN1, MPL, MSN, MTCP1, MUC1, MUTYH, MYCL(MYCL1), NBN, NDRG1, NKX2-1, NONO, NOTCH1, NRAS, NUMA1, NUTM2B, OLIG2,OMD, P2RY8, PAFAH1B2, PAK3, PATZ1, PAX8, PDE4DIP, PHF6, PHOX2B, PIK3CG,PLAG1, PMS1, POU5F1, PPP2R1A, PRF1, PRKDC, RAD21, RECQL4, RHOH, RNF213,RPL10, SEPT5, SEPT6, SFPQ, SLC45A3, SMARCA4, SOCS1, SOX2, SPOP, SRC,SSX1, STAG2, TAL1, TAL2, TBL1XR1, TCEA1, TCL1A, TERT, TFE3, TFPT,THRAP3, TLX3, TMPRSS2, UBR5, VHL, WAS, ZBTB16, and ZRSR2.

In the methods of the invention, including enriching an oligonucleotidelibrary, characterizing a sample or visualizing a sample, the phenotypecan be a phenotype comprises a disease or disorder. The methods can beemployed to assist in providing a diagnosis, prognosis and/or theranosisfor the disease or disorder. For example, the enriching may be performedusing sample such that the enriched library can be used to assist inproviding a diagnosis, prognosis and/or theranosis for the disease ordisorder. Similarly, the characterizing may comprise assisting inproviding a diagnosis, prognosis and/or theranosis for the disease ordisorder. The visualization may also comprise assisting in providing adiagnosis, prognosis and/or theranosis for the disease or disorder. Insome embodiments, the theranosis comprises predicting a treatmentefficacy or lack thereof, classifying a patient as a responder ornon-responder to treatment, or monitoring a treatment efficacy. Thetheranosis can be directed to any appropriate treatment, e.g., thetreatment may comprise at least one of chemotherapy, immunotherapy,targeted cancer therapy, a monoclonal antibody, an anti-HER2 antibody,trastuzumab, an anti-VEGF antibody, bevacizumab, and/or platinum/taxanetherapy. In some embodiments, the treatment comprises at least one ofafatinib, afatinib+cetuximab, alectinib, aspirin, atezolizumab,bicalutamide, cabozantinib, capecitabine, carboplatin, ceritinib,cetuximab, cisplatin, crizotinib, dabrafenib, dacarbazine, doxorubicin,enzalutamide, epirubicin, erlotinib, everolimus, exemestane+everolimus,fluorouracil, fulvestrant, gefitinib, gemcitabine, hormone therapies,irinotecan, lapatinib, liposomal-doxorubicin, matinib, mitomycin-c,nab-paclitaxel, nivolumab, olaparib, osimertinib, oxaliplatin,palbociclib combination therapy, paclitaxel, palbociclib, panitumumab,pembrolizumab, pemetrexed, pertuzumab, sunitinib, T-DM1, temozolomidedocetaxel, temsirolimus, topotecan, trametinib, trastuzumab, vandetanib,and vemurafenib. The hormone therapy can be one or more of tamoxifen,toremifene, fulvestrant, letrozole, anastrozole, exemestane, megestrolacetate, leuprolide, goserelin, bicalutamide, flutamide, abiraterone,enzalutamide, triptorelin, abarelix, and degarelix.

In the methods of the invention directed to characterizing a sample, thecharacterizing may comprise comparing the presence or level to areference. In some embodiments, the reference comprises a presence orlevel determined in a sample from an individual without a disease ordisorder, or from an individual with a different state of a disease ordisorder. The presence or level can be that of a visual level, such asan IHC score, determined by the visualizing. As a non-limiting example,the comparison to the reference of at least one oligonucleotide orplurality of oligonucleotides provided by the invention indicates thatthe sample comprises a cancer sample or a non-cancer/normal sample.

In some embodiments of the methods of the invention, one or more samplecomprises a bodily fluid. The bodily fluid can be any useful bodilyfluid, including without limitation peripheral blood, sera, plasma,ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow,synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk,broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid orpre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair oil,tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid,lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum,vomit, vaginal secretions, mucosal secretion, stool water, pancreaticjuice, lavage fluids from sinus cavities, bronchopulmonary aspirates,blastocyl cavity fluid, or umbilical cord blood.

In the methods of the invention, including characterizing a sample orvisualizing a sample, the sample can be from a subject suspected ofhaving or being predisposed to a medical condition, disease, ordisorder.

In the methods of the invention, including enriching an oligonucleotidelibrary, characterizing a sample or visualizing a sample, the medicalcondition, the disease or disorder may be a cancer, a premalignantcondition, an inflammatory disease, an immune disease, an autoimmunedisease or disorder, a cardiovascular disease or disorder, neurologicaldisease or disorder, infectious disease or pain. In some embodiments,the cancer comprises an acute lymphoblastic leukemia; acute myeloidleukemia; adrenocortical carcinoma; AIDS-related cancers; AIDS-relatedlymphoma; anal cancer; appendix cancer; astrocytomas; atypicalteratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer; brainstem glioma; brain tumor (including brain stem glioma, central nervoussystem atypical teratoid/rhabdoid tumor, central nervous systemembryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma,ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymaltumors of intermediate differentiation, supratentorial primitiveneuroectodermal tumors and pineoblastoma); breast cancer; bronchialtumors; Burkitt lymphoma; cancer of unknown primary site; carcinoidtumor; carcinoma of unknown primary site; central nervous systematypical teratoid/rhabdoid tumor; central nervous system embryonaltumors; cervical cancer; childhood cancers; chordoma; chroniclymphocytic leukemia; chronic myelogenous leukemia; chronicmyeloproliferative disorders; colon cancer; colorectal cancer;craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas isletcell tumors; endometrial cancer; ependymoblastoma; ependymoma;esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranialgerm cell tumor; extragonadal germ cell tumor; extrahepatic bile ductcancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinalcarcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinalstromal tumor (GIST); gestational trophoblastic tumor; glioma; hairycell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma;hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposisarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer;lip cancer; liver cancer; lung cancer; malignant fibrous histiocytomabone cancer; medulloblastoma; medulloepithelioma; melanoma; Merkel cellcarcinoma; Merkel cell skin carcinoma; mesothelioma; metastatic squamousneck cancer with occult primary; mouth cancer; multiple endocrineneoplasia syndromes; multiple myeloma; multiple myeloma/plasma cellneoplasm; mycosis fungoides; myelodysplastic syndromes;myeloproliferative neoplasms; nasal cavity cancer; nasopharyngealcancer; neuroblastoma; Non-Hodgkin lymphoma; nonmelanoma skin cancer;non-small cell lung cancer; oral cancer; oral cavity cancer;oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors;ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor;ovarian low malignant potential tumor; pancreatic cancer;papillomatosis; paranasal sinus cancer; parathyroid cancer; pelviccancer; penile cancer; pharyngeal cancer; pineal parenchymal tumors ofintermediate differentiation; pineoblastoma; pituitary tumor; plasmacell neoplasm/multiple myeloma; pleuropulmonary blastoma; primarycentral nervous system (CNS) lymphoma; primary hepatocellular livercancer; prostate cancer; rectal cancer; renal cancer; renal cell(kidney) cancer; renal cell cancer; respiratory tract cancer;retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sézarysyndrome; small cell lung cancer; small intestine cancer; soft tissuesarcoma; squamous cell carcinoma; squamous neck cancer; stomach(gastric) cancer; supratentorial primitive neuroectodermal tumors;T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma;thymoma; thyroid cancer; transitional cell cancer; transitional cellcancer of the renal pelvis and ureter; trophoblastic tumor; uretercancer; urethral cancer; uterine cancer; uterine sarcoma; vaginalcancer; vulvar cancer; Waldenström macroglobulinemia; or Wilm's tumor.In some embodiments, the premalignant condition comprises Barrett'sEsophagus. In some embodiments, the autoimmune disease comprisesinflammatory bowel disease (IBD), Crohn's disease (CD), ulcerativecolitis (UC), pelvic inflammation, vasculitis, psoriasis, diabetes,autoimmune hepatitis, multiple sclerosis, myasthenia gravis, Type Idiabetes, rheumatoid arthritis, psoriasis, systemic lupus erythematosis(SLE), Hashimoto's Thyroiditis, Grave's disease, Ankylosing SpondylitisSjogrens Disease, CREST syndrome, Scleroderma, Rheumatic Disease, organrejection, Primary Sclerosing Cholangitis, or sepsis. In someembodiments, the cardiovascular disease comprises atherosclerosis,congestive heart failure, vulnerable plaque, stroke, ischemia, highblood pressure, stenosis, vessel occlusion or a thrombotic event. Insome embodiments, the neurological disease comprises Multiple Sclerosis(MS), Parkinson's Disease (PD), Alzheimer's Disease (AD), schizophrenia,bipolar disorder, depression, autism, Prion Disease, Pick's disease,dementia, Huntington disease (HD), Down's syndrome, cerebrovasculardisease, Rasmussen's encephalitis, viral meningitis, neurospsychiatricsystemic lupus erythematosus (NPSLE), amyotrophic lateral sclerosis,Creutzfeldt-Jacob disease, Gerstmann-Straussler-Scheinker disease,transmissible spongiform encephalopathy, ischemic reperfusion damage(e.g. stroke), brain trauma, microbial infection, or chronic fatiguesyndrome. In some embodiments, the pain comprises fibromyalgia, chronicneuropathic pain, or peripheral neuropathic pain. In some embodiments,the infectious disease comprises a bacterial infection, viral infection,yeast infection, Whipple's Disease, Prion Disease, cirrhosis,methicillin-resistant Staphylococcus aureus, HIV, HCV, hepatitis,syphilis, meningitis, malaria, tuberculosis, influenza.

In an aspect, the invention provides a kit comprising at least onereagent for carrying out the methods provided by the invention,including enriching an oligonucleotide library, characterizing a sampleor visualizing a sample. In a related aspect, the invention provides useof at least one reagent for carrying out the methods provided by theinvention, including enriching an oligonucleotide library,characterizing a sample or visualizing a sample. In some embodiments,the at least one reagent comprises an oligonucleotide or a plurality ofoligonucleotides provided herein. Additional useful reagents are alsoprovided herein. See, e.g., the protocols provided in the Examples.

In an aspect, the invention provides a method of imaging at least onecell or tissue, comprising contacting the at least one cell or tissuewith at least one oligonucleotide or plurality of oligonucleotidesprovided herein, and detecting the at least one oligonucleotide or theplurality of oligonucleotides in contact with at least one cell ortissue.

For example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids may have a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or allof SEQ ID NOs. 2922-2926, 2929-2947 and 2950-2952. In such cases, theimaging may be, e.g., directed to lung or prostate tissue.

In another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or allof SEQ ID NOs. 2953-2961 and 2971-2979. In such cases, the phenotype maybe, e.g., prostate cancer.

In yet another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50 or all of SEQ ID NOs. 3039-3061. In such cases, the imagingmay be, e.g., directed to HER2 status of a cell or tissue.

In still another example, the at least one oligonucleotide or pluralityof oligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 150,000 or allof SEQ ID NOs. 3062-103061 and 103062-203061. In such cases, the imagingmay be, e.g., directed to a HER2 status of a cell or tissue.

In an example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000 or all of SEQ ID NOs. 203064-203067 and203076-206478. In such cases, the imaging may be, e.g., directed tocolorectal cells or tissue.

In another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or all ofSEQ ID NOs. 206491-206506. In such cases, the imaging may be, e.g.,directed to a tissue, including without limitation breast, colon,kidney, lung or pancreatic tissue.

In the imaging methods provided by the invention, the at least oneoligonucleotide or the plurality of oligonucleotides can carry varioususeful chemical structures or modifications such as described herein.

In the imaging methods provided by the invention, the at least oneoligonucleotide or the plurality of oligonucleotides can be administeredto a subject prior to the detecting. Such method may allow imaging of atleast one cell or tissue in the subject. In some embodiments, the atleast one cell or tissue comprises neoplastic, malignant, tumor,hyperplastic, or dysplastic cells. In some embodiments, the at least onecell or tissue comprises at least one of lymphoma, leukemia, renalcarcinoma, sarcoma, hemangiopericytoma, melanoma, abdominal cancer,gastric cancer, colon cancer, cervical cancer, prostate cancer,pancreatic cancer, breast cancer, or non-small cell lung cancer cells.In some embodiments, the at least one cell or tissue comprises a medicalcondition, disease or disorder.

In an aspect, the invention provides a pharmaceutical compositioncomprising a construct comprising a therapeutically effective amount ofthe at least one oligonucleotide or the plurality of oligonucleotides asprovided herein, or a salt thereof, and a pharmaceutically acceptablecarrier, diluent, or both. In some embodiments, the at least oneoligonucleotide or the plurality of oligonucleotides associates with atleast one protein listed in Table 28 herein.

For example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or allof SEQ ID NOs. 2922-2926, 2929-2947 and 2950-2952. Such pharmaceuticalcomposition may be useful for therapy related to a cancer, whereinoptionally the cancer comprises lung cancer or prostate cancer.

In another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or allof SEQ ID NOs. 2953-2961 and 2971-2979. Such pharmaceutical compositionmay be useful for therapy related to a cancer, wherein optionally thecancer comprises prostate cancer.

In still another example, the at least one oligonucleotide or pluralityof oligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50 or all of SEQ ID NOs. 3039-3061. Such pharmaceuticalcomposition may be useful for therapy related to a cancer, whereinoptionally the cancer comprises breast cancer.

In yet another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 150,000 or allof SEQ ID NOs. 3062-103061 and 103062-203061. Such pharmaceuticalcomposition may be useful for therapy related to a cancer, whereinoptionally the cancer comprises breast cancer.

In an example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000 or all of SEQ ID NOs. 203064-203067 and203076-206478. Such pharmaceutical composition may be useful for therapyrelated to a cancer, wherein optionally the cancer comprises colorectalcancer.

In yet another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or all ofSEQ ID NOs. 206491-206506. Such pharmaceutical composition may be usefulfor therapy related to a cancer, wherein optionally the cancer comprisesa cancer of the breast, colon, kidney, lung or pancreas.

The at least one oligonucleotide or the plurality of oligonucleotideswithin the pharmaceutical composition can have any useful desiredchemical modification. In an embodiment, the at least oneoligonucleotide or the plurality of oligonucleotides is attached to atoxin or chemotherapeutic agent. The at least one oligonucleotide or theplurality of oligonucleotides may be comprised within a multipartiteconstruct. The at least one oligonucleotide or the plurality ofoligonucleotides can be attached to a liposome or nanoparticle. In someembodiments, the liposome or nanoparticle comprises a toxin orchemotherapeutic agent.

In a related aspect, the invention provides a method of treating orameliorating a disease or disorder in a subject in need thereof,comprising administering the pharmaceutical composition of the inventionto the subject. In another related aspect, the invention provides amethod of inducing cytotoxicity in a subject, comprising administeringthe pharmaceutical composition of the invention to the subject. Anyuseful means of administering can be used, including without limitationat least one of intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, oral, sublingual,intracerebral, intravaginal, transdermal, rectal, by inhalation, topicaladministration, or any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B illustrate methods of assessing biomarkers such as cellularor microvesicle surface antigens. FIG. 1A is a schematic of a planarsubstrate coated with a capture agent, such as an aptamer or antibody,which captures cells or microvesicles expressing the target antigen ofthe capture agent. The capture agent may bind a protein expressed on thesurface of the diseased cell or vesicle. The detection agent, which mayalso be an aptamer or antibody, carries a detectable label, here afluorescent signal. The detection agent binds to the captured cell ormicrovesicle and provides a detectable signal via its fluorescent label.The detection agent can detect an antigen that is generally associated acell-of-origin or a disease, e.g., a cancer. FIG. 1B is a schematic of aparticle bead conjugated with a capture agent, which captures cells ormicrovesicles expressing the target antigen of the capture agent. Thecapture agent may bind a protein expressed on the surface of thediseased cell or vesicle. The detection agent, which may also be anaptamer or antibody, carries a detectable label, here a fluorescentsignal. The detection agent binds to the captured cell or microvesicleand provides a detectable signal via its fluorescent label. Thedetection agent can detect an antigen that is generally associated witha cell-of-origin or a disease, e.g., a cancer.

FIGS. 2A-B illustrates a non-limiting example of an aptamer nucleotidesequence and its secondary structure. FIG. 2A illustrates a secondarystructure of a 32-mer oligonucleotide, Aptamer 4, with sequence5′-CCCCCCGAATCACATGACTTGGGCGGGGGTCG (SEQ ID NO. 1). In the figure, thesequence is shown with 6 thymine nucleotides added to the end, which canact as a spacer to attach a biotin molecule. This particular oligo has ahigh binding affinity to the target, EpCAM. Additional candidate EpCAMbinders are identified by modeling the entire database of sequencedoligos to the secondary structure of this oligo. FIG. 2B illustratesanother 32-mer oligo with sequence 5′-ACCGGATAGCGGTTGGAGGCGTGCTCCACTCG(SEQ ID NO. 2) that has a different secondary structure than the aptamerin FIG. 2A. This aptamer is also shown with a 6-thymine tail.

FIG. 3 illustrates a process for producing a target-specific set ofaptamers using a cell subtraction method, wherein the target is abiomarker associated with a specific disease. In Step 1, a random poolof oligonucleotides are contacted with a biological sample from a normalpatient. In Step 2, the oligos that did not bind in Step 1 are added toa biological sample isolated from diseased patients. The bound oligosfrom this step are then eluted, captured via their biotin linkage andthen combined again with normal biological sample. The unbound oligosare then added again to disease-derived biological sample and isolated.This process can be repeated iteratively. The final eluted aptamers aretested against patient samples to measure the sensitivity andspecificity of the set. Biological samples can include blood, includingplasma or serum, or other components of the circulatory system, such asmicrovesicles.

FIG. 4 comprises a schematic for identifying a target of a selectedaptamer, such as an aptamer selected by the process of the invention.The figure shows a binding agent 402, here an aptamer for purposes ofillustration, tethered to a substrate 401. The binding agent 402 can becovalently attached to substrate 401. The binding agent 402 may also benon-covalently attached. For example, binding agent 402 can comprise alabel which can be attracted to the substrate, such as a biotin groupwhich can form a complex with an avidin/streptavidin molecule that iscovalently attached to the substrate. The binding agent 402 binds to asurface antigen 403 of microvesicle 404. In the step signified by arrow(i), the microvesicle is disrupted while leaving the complex between thebinding agent 402 and surface antigen 403 intact. Disrupted microvesicle405 is removed, e.g., via washing or buffer exchange, in the stepsignified by arrow (ii). In the step signified by arrow (iii), thesurface antigen 403 is released from the binding agent 402. The surfaceantigen 403 can be analyzed to determine its identity.

FIGS. 5A-5G illustrate using an oligonucleotide probe library todifferentiate cancer and non-cancer samples.

FIG. 6 shows protein targets of oligonucleotide probes run on a silverstained SDS-PAGE gel.

FIGS. 7A-B illustrate a model generated using a training (FIG. 7A) andtest (FIG. 7B) set from a round of cross validation. The AUC for thetest set was 0.803. Another exemplary round of cross-validation is shownin FIGS. 7C-D with training (FIG. 7C) and test (FIG. 7D) sets. The AUCfor the test set was 0.678.

FIG. 8 illustrates multipart oligonucleotide constructs.

FIGS. 9A-C illustrate SUPRA (SsDNA by Unequal length PRimer AsymmetricPCR), a protocol for single stranded DNA (ssDNA) oligonucleotide librarypreparation.

FIGS. 10A-D illustrate use of aptamers in methods of characterizing aphenotype. FIG. 10A is a schematic 1000 showing an assay configurationthat can be used to detect and/or quantify a target of interest. In thefigure, capture aptamer 1002 is attached to substrate 1001. Target ofinterest 1003 is bound by capture aptamer 1002. Detection aptamer 1004is also bound to target of interest 1003. Detection aptamer 1004 carrieslabel 1005 which can be detected to identify target captured tosubstrate 1001 via capture aptamer 1002. FIG. 10B is a schematic 1010showing use of an aptamer pool to characterize a phenotype. A pool ofaptamers to a target of interest is provided 1011. The pool is contactedwith a test sample to be characterized 1012. The mixture is washed toremove unbound aptamers. The remaining aptamers are disassociated andcollected 1013. The collected aptamers are identified 1014 and theidentity of the retained aptamers is used to characterize the phenotype1015. FIG. 10C is a schematic 1020 showing an implementation of themethod in FIG. 10B. A pool of aptamers identified as binding amicrovesicle population is provided 1019. The input sample comprisesmicrovesicles that are isolated from a test sample 1020. The pool iscontacted with the isolated microvesicles to be characterized 1023. Themixture is washed to remove unbound aptamers and the remaining aptamersare disassociated and collected 1025. The collected aptamers areidentified and the identity of the retained aptamers is used tocharacterize the phenotype 1026. FIG. 10D provides an outline 1030 ofsuch method. An aptamer pool is provided that has been enriched againsta tissue of interest 1031. The pool is contacted with a tissue sample1032. The tissue sample can be in a format such as described herein. Asa non-limiting example, the tissue sample can be a fixed tumor sample.The sample may be a FFPE sample fixed to a glass slide or membrane. Thesample is washed to remove unbound members of the aptamer pool and theremaining aptamers are visualized 1033. Any appropriate method tovisualize the aptamers can be used. In an example, the aptamer pool isbiotinylated and the bound aptamer are visualized usingstreptavidin-horse radish peroxidase (SA-HRP). As described herein,other useful visualization methods are known in the art, includingalternate labeling. The visualized sample is scored to determine theamount of staining 1034. For example a pathologist can score the slideas in IHC. The score can be used to characterize the sample 1035 asdescribed herein. For example, a score of +1 or higher may indicate thatthe sample is a cancer sample, or is a cancer sample expressing a givenbiomarker.

FIGS. 11A-I illustrate development and use of an oligonucleotide probelibrary to distinguish biological sample types.

FIGS. 12A-C illustrate enriching a naïve oligonucleotide library withbalanced design for oligonucleotides that differentiate between breastcancer and non-cancer microvesicles derived from plasma samples.

FIG. 13 shows a schematic for enriching an oligonucleotide libraryagainst cell lines.

FIGS. 14A-C show oligonucleotide probes that recognize microvesicles(exosomes) shed by prostate cancer cell lines.

FIGS. 15A-E show identification of oligonucleotide probes that recognizeHER2+ cancer samples.

FIGS. 16A-F show oligonucleotide probes that distinguish trastuzamabresponder breast cancer samples.

FIGS. 17A-P show oligonucleotide probes that distinguish trastuzamabresponder breast cancer tissue samples.

FIGS. 18A-G show development of oligonucleotide probes that predict theresponse to a combinational therapy with FOLFOX/bevacizumab inindividuals diagnosed with colorectal cancer.

FIGS. 19A-K show background optimization of oligonucleotide probestaining of fixed tissue samples.

FIGS. 20A-D show oligonucleotide probes that distinguish tubulin 3(TUBB3) positive and negative pancreatic cancer tissue samples.

FIGS. 21A-B show development of oligonucleotide probes that predict theresponse to platinum/taxane therapy in individuals diagnosed withovarian cancer.

FIGS. 22A-B show enrichment and staining of an oligonucleotide probelibrary against kidney tissue anti-digoxigenin (DIG) antibody detection.

FIGS. 23A-D illustrate oligonucleotide probe library enrichment usinglysates from fixed tissue samples.

FIGS. 24A-B illustrate oligonucleotide probe library enrichment usingscraped tissue from cancer and non-cancer regions from fixed tissuesamples.

FIGS. 25A-B illustrate oligonucleotide probe library enrichment usingmicrodissection of fixed tissue samples.

FIGS. 26A-B illustrate therapeutic agents whose benefit or lack ofbenefit for treating a cancer may depend on a biomarker status.

DETAILED DESCRIPTION OF THE INVENTION

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. Other features, objects, and advantages ofthe invention will be apparent from the description. In thespecification, the singular forms also include the plural unless thecontext clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. In the case of conflict, the present Specificationwill control.

Disclosed herein are compositions and methods that can be used tocharacterize a phenotype, or assess, a biological sample. Thecompositions and methods of the invention comprise the use ofoligonucleotide probes (aptamers) that bind biological entities ofinterest, including without limitation tissues, cell, microvesicles, orfragments thereof. The antigens recognized by the oligonucleotideaptamers may comprise proteins or polypeptides or any other usefulbiological components such as nucleic acids, lipids and/orcarbohydrates. In general, the oligonucleotides disclosed are syntheticnucleic acid molecules, including DNA and RNA, and variations thereof.Unless otherwise specified, the oligonucleotide probes can besynthesized in DNA or RNA format or as hybrid molecules as desired. Themethods disclosed herein comprise diagnostic, prognostic and theranosticprocesses and techniques using one or more aptamer of the invention.Alternatively, an oligonucleotide probe of the invention can also beused as a binding agent to capture, isolate, or enrich, a cell, cellfragment, microvesicle or any other fragment or complex that comprisesthe antigen or functional fragments thereof.

The compositions and methods of the invention also comprise individualoligonucleotides that can be used to assess biological samples. Theinvention further discloses compositions and methods of oligonucleotidepools that can be used to detect a biosignature in a sample.

Oligonucleotide probes and sequences disclosed in the compositions andmethods of the invention may be identified herein in the form of DNA orRNA. Unless otherwise specified, one of skill in the art will appreciatethat an oligonucleotide may generally be synthesized as either form ofnucleic acid and carry various chemical modifications and remain withinthe scope of the invention. The term aptamer may be used in the art torefer to a single oligonucleotide that binds specifically to a target ofinterest through mechanisms other than Watson crick base pairing,similar to binding of a monoclonal antibody to a particular antigen.Within the scope of this disclosure and unless stated explicitly orotherwise implicit in context, the terms aptamer, oligonucleotide andoligonucleotide probe, and variations thereof, may be usedinterchangeably to refer to an oligonucleotide capable of distinguishingbiological entities of interest (e.g, tissues, cells, microvesicles,biomarkers) whether or not the specific entity has been identified orwhether the precise mode of binding has been determined.

An oligonucleotide probe or plurality of such probes of the inventioncan also be used to provide in vitro or in vivo detection or imaging andto provide diagnostic readouts, including for diagnostic, prognostic ortheranostic purposes.

Separately, an oligonucleotide probe of the invention can also be usedfor treatment or as a therapeutic to specifically target a cell, tissue,organ or the like. As the invention provides methods to identifyoligonucleotide probes that bind to specific tissues, cells,microvesicles or other biological entities of interest, theoligonucleotide probes of the invention target such entities and areinherently drug candidates, agents that can be used for targeted drugdelivery, or both.

Phenotypes

Disclosed herein are products and processes for characterizing aphenotype using the methods and compositions of the invention. The term“phenotype” as used herein can mean any trait or characteristic that canbe identified using in part or in whole the compositions and/or methodsof the invention. For example, a phenotype can be a diagnostic,prognostic or theranostic determination based on a characterizedbiomarker profile for a sample obtained from a subject. A phenotype canbe any observable characteristic or trait of, such as a disease orcondition, a stage of a disease or condition, susceptibility to adisease or condition, prognosis of a disease stage or condition, aphysiological state, or response/potential response to therapeutics. Aphenotype can result from a subject's genetic makeup as well as theinfluence of environmental factors and the interactions between the two,as well as from epigenetic modifications to nucleic acid sequences.

A phenotype in a subject can be characterized by obtaining a biologicalsample from a subject and analyzing the sample using the compositionsand/or methods of the invention. For example, characterizing a phenotypefor a subject or individual can include detecting a disease or condition(including pre-symptomatic early stage detecting), determining aprognosis, diagnosis, or theranosis of a disease or condition, ordetermining the stage or progression of a disease or condition.Characterizing a phenotype can include identifying appropriatetreatments or treatment efficacy for specific diseases, conditions,disease stages and condition stages, predictions and likelihood analysisof disease progression, particularly disease recurrence, metastaticspread or disease relapse. A phenotype can also be a clinically distincttype or subtype of a condition or disease, such as a cancer or tumor.Phenotype determination can also be a determination of a physiologicalcondition, or an assessment of organ distress or organ rejection, suchas post-transplantation. The compositions and methods described hereinallow assessment of a subject on an individual basis, which can providebenefits of more efficient and economical decisions in treatment.

In an aspect, the invention relates to the analysis of tissues,microvesicles, and circulating biomarkers to provide a diagnosis,prognosis, and/or theranosis of a disease or condition. Theranosticsincludes diagnostic testing that provides the ability to affect therapyor treatment of a disease or disease state. Theranostics testingprovides a theranosis in a similar manner that diagnostics or prognostictesting provides a diagnosis or prognosis, respectively. As used herein,theranostics encompasses any desired form of therapy related testing,including predictive medicine, personalized medicine, precisionmedicine, integrated medicine, pharmacodiagnostics and Dx/Rx partnering.Therapy related tests can be used to predict and assess drug response inindividual subjects, i.e., to provide personalized medicine. Predictinga drug response can be determining whether a subject is a likelyresponder or a likely non-responder to a candidate therapeutic agent,e.g., before the subject has been exposed or otherwise treated with thetreatment. Assessing a drug response can be monitoring a response to adrug, e.g., monitoring the subject's improvement or lack thereof over atime course after initiating the treatment. Therapy related tests areuseful to select a subject for treatment who is particularly likely tobenefit from the treatment or to provide an early and objectiveindication of treatment efficacy in an individual subject. Thus,analysis using the compositions and methods of the invention mayindicate that treatment should be altered to select a more promisingtreatment, thereby avoiding the great expense of delaying beneficialtreatment and avoiding the financial and morbidity costs ofadministering an ineffective drug(s).

In assessing a phenotype, a biosignature can be analyzed in the subjectand compared against that of previous subjects that were known torespond or not to a treatment. The biosignature may comprise certainbiomarkers or may comprise certain detection agents, such as theoligonucleotide probes as provided herein. If the biosignature in thesubject more closely aligns with that of previous subjects that wereknown to respond to the treatment, the subject can be characterized, orpredicted, as a responder to the treatment. Similarly, if the biomarkerprofile in the subject more closely aligns with that of previoussubjects that did not respond to the treatment, the subject can becharacterized, or predicted as a non-responder to the treatment. Thetreatment can be for any appropriate disease, disorder or othercondition, including without limitation those disclosed herein.

In some embodiments, the phenotype comprises a medical conditionincluding without limitation a disease or disorder listed in Table 1.For example, the phenotype can comprise detecting the presence of orlikelihood of developing a tumor, neoplasm, or cancer, or characterizingthe tumor, neoplasm, or cancer (e.g., stage, grade, aggressiveness,likelihood of metastatis or recurrence, etc). Cancers that can bedetected or assessed by methods or compositions described hereininclude, but are not limited to, breast cancer, ovarian cancer, lungcancer, colon cancer, hyperplastic polyp, adenoma, colorectal cancer,high grade dysplasia, low grade dysplasia, prostatic hyperplasia,prostate cancer, melanoma, pancreatic cancer, brain cancer (such as aglioblastoma), hematological malignancy, hepatocellular carcinoma,cervical cancer, endometrial cancer, head and neck cancer, esophagealcancer, gastrointestinal stromal tumor (GIST), renal cell carcinoma(RCC) or gastric cancer. The colorectal cancer can be CRC Dukes B orDukes C-D. The hematological malignancy can be B-Cell ChronicLymphocytic Leukemia, B-Cell Lymphoma-DLBCL, B-CellLymphoma-DLBCL-germinal center-like, B-Cell Lymphoma-DLBCL-activatedB-cell-like, and Burkitt's lymphoma.

The phenotype can be a premalignant condition, such as actinickeratosis, atrophic gastritis, leukoplakia, erythroplasia, LymphomatoidGranulomatosis, preleukemia, fibrosis, cervical dysplasia, uterinecervical dysplasia, xeroderma pigmentosum, Barrett's Esophagus,colorectal polyp, or other abnormal tissue growth or lesion that islikely to develop into a malignant tumor. Transformative viralinfections such as HIV and HPV also present phenotypes that can beassessed according to the invention.

A cancer characterized by the compositions and methods of the inventioncan comprise, without limitation, a carcinoma, a sarcoma, a lymphoma orleukemia, a germ cell tumor, a blastoma, or other cancers. Carcinomasinclude without limitation epithelial neoplasms, squamous cell neoplasmssquamous cell carcinoma, basal cell neoplasms basal cell carcinoma,transitional cell papillomas and carcinomas, adenomas andadenocarcinomas (glands), adenoma, adenocarcinoma, linitis plasticainsulinoma, glucagonoma, gastrinoma, vipoma, cholangiocarcinoma,hepatocellular carcinoma, adenoid cystic carcinoma, carcinoid tumor ofappendix, prolactinoma, oncocytoma, hurthle cell adenoma, renal cellcarcinoma, grawitz tumor, multiple endocrine adenomas, endometrioidadenoma, adnexal and skin appendage neoplasms, mucoepidermoid neoplasms,cystic, mucinous and serous neoplasms, cystadenoma, pseudomyxomaperitonei, ductal, lobular and medullary neoplasms, acinar cellneoplasms, complex epithelial neoplasms, warthin's tumor, thymoma,specialized gonadal neoplasms, sex cord stromal tumor, thecoma,granulosa cell tumor, arrhenoblastoma, sertoli leydig cell tumor, glomustumors, paraganglioma, pheochromocytoma, glomus tumor, nevi andmelanomas, melanocytic nevus, malignant melanoma, melanoma, nodularmelanoma, dysplastic nevus, lentigo maligna melanoma, superficialspreading melanoma, and malignant acral lentiginous melanoma. Sarcomaincludes without limitation Askin's tumor, botryodies, chondrosarcoma,Ewing's sarcoma, malignant hemangio endothelioma, malignant schwannoma,osteosarcoma, soft tissue sarcomas including: alveolar soft partsarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma,desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma,extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma,hemangiopericytoma, hemangiosarcoma, kaposi's sarcoma, leiomyosarcoma,liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibroushistiocytoma, neurofibrosarcoma, rhabdomyosarcoma, and synovialsarcoma.Lymphoma and leukemia include without limitation chronic lymphocyticleukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia,lymphoplasmacytic lymphoma (such as waldenstrom macroglobulinemia),splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma,monoclonal immunoglobulin deposition diseases, heavy chain diseases,extranodal marginal zone B cell lymphoma, also called malt lymphoma,nodal marginal zone B cell lymphoma (nmzl), follicular lymphoma, mantlecell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) largeB cell lymphoma, intravascular large B cell lymphoma, primary effusionlymphoma, burkitt lymphoma/leukemia, T cell prolymphocytic leukemia, Tcell large granular lymphocytic leukemia, aggressive NK cell leukemia,adult T cell leukemia/lymphoma, extranodal NK/T cell lymphoma, nasaltype, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma,blastic NK cell lymphoma, mycosis fungoides/sezary syndrome, primarycutaneous CD30-positive T cell lymphoproliferative disorders, primarycutaneous anaplastic large cell lymphoma, lymphomatoid papulosis,angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma,unspecified, anaplastic large cell lymphoma, classical hodgkin lymphomas(nodular sclerosis, mixed cellularity, lymphocyte-rich, lymphocytedepleted or not depleted), and nodular lymphocyte-predominant hodgkinlymphoma. Germ cell tumors include without limitation germinoma,dysgerminoma, seminoma, nongerminomatous germ cell tumor, embryonalcarcinoma, endodermal sinus turmor, choriocarcinoma, teratoma,polyembryoma, and gonadoblastoma. Blastoma includes without limitationnephroblastoma, medulloblastoma, and retinoblastoma. Other cancersinclude without limitation labial carcinoma, larynx carcinoma,hypopharynx carcinoma, tongue carcinoma, salivary gland carcinoma,gastric carcinoma, adenocarcinoma, thyroid cancer (medullary andpapillary thyroid carcinoma), renal carcinoma, kidney parenchymacarcinoma, cervix carcinoma, uterine corpus carcinoma, endometriumcarcinoma, chorion carcinoma, testis carcinoma, urinary carcinoma,melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma,medulloblastoma and peripheral neuroectodermal tumors, gall bladdercarcinoma, bronchial carcinoma, multiple myeloma, basalioma, teratoma,retinoblastoma, choroidea melanoma, seminoma, rhabdomyosarcoma,craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma,liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmocytoma.

In a further embodiment, the cancer under analysis may be a lung cancerincluding non-small cell lung cancer and small cell lung cancer(including small cell carcinoma (oat cell cancer), mixed smallcell/large cell carcinoma, and combined small cell carcinoma), coloncancer, breast cancer, prostate cancer, liver cancer, pancreas cancer,brain cancer, kidney cancer, ovarian cancer, stomach cancer, skincancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer,glioma, glioblastoma, hepatocellular carcinoma, papillary renalcarcinoma, head and neck squamous cell carcinoma, leukemia, lymphoma,myeloma, or a solid tumor.

In embodiments, the cancer comprises an acute lymphoblastic leukemia;acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancers;AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas;atypical teratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer;brain stem glioma; brain tumor (including brain stem glioma, centralnervous system atypical teratoid/rhabdoid tumor, central nervous systemembryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma,ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymaltumors of intermediate differentiation, supratentorial primitiveneuroectodermal tumors and pineoblastoma); breast cancer; bronchialtumors; Burkitt lymphoma; cancer of unknown primary site; carcinoidtumor; carcinoma of unknown primary site; central nervous systematypical teratoid/rhabdoid tumor; central nervous system embryonaltumors; cervical cancer; childhood cancers; chordoma; chroniclymphocytic leukemia; chronic myelogenous leukemia; chronicmyeloproliferative disorders; colon cancer; colorectal cancer;craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas isletcell tumors; endometrial cancer; ependymoblastoma; ependymoma;esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranialgerm cell tumor; extragonadal germ cell tumor; extrahepatic bile ductcancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinalcarcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinalstromal tumor (GIST); gestational trophoblastic tumor; glioma; hairycell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma;hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposisarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer;lip cancer; liver cancer; malignant fibrous histiocytoma bone cancer;medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma;Merkel cell skin carcinoma; mesothelioma; metastatic squamous neckcancer with occult primary; mouth cancer; multiple endocrine neoplasiasyndromes; multiple myeloma; multiple myeloma/plasma cell neoplasm;mycosis fungoides; myelodysplastic syndromes; myeloproliferativeneoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma;Non-Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lungcancer; oral cancer; oral cavity cancer; oropharyngeal cancer;osteosarcoma; other brain and spinal cord tumors; ovarian cancer;ovarian epithelial cancer; ovarian germ cell tumor; ovarian lowmalignant potential tumor; pancreatic cancer; papillomatosis; paranasalsinus cancer; parathyroid cancer; pelvic cancer; penile cancer;pharyngeal cancer; pineal parenchymal tumors of intermediatedifferentiation; pineoblastoma; pituitary tumor; plasma cellneoplasm/multiple myeloma; pleuropulmonary blastoma; primary centralnervous system (CNS) lymphoma; primary hepatocellular liver cancer;prostate cancer; rectal cancer; renal cancer; renal cell (kidney)cancer; renal cell cancer; respiratory tract cancer; retinoblastoma;rhabdomyosarcoma; salivary gland cancer; Sézary syndrome; small celllung cancer; small intestine cancer; soft tissue sarcoma; squamous cellcarcinoma; squamous neck cancer; stomach (gastric) cancer;supratentorial primitive neuroectodermal tumors; T-cell lymphoma;testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroidcancer; transitional cell cancer; transitional cell cancer of the renalpelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer;uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer;Waldenström macroglobulinemia; or Wilm's tumor. The methods of theinvention can be used to characterize these and other cancers. Thus,characterizing a phenotype can be providing a diagnosis, prognosis ortheranosis of one of the cancers disclosed herein.

In some embodiments, the cancer comprises an acute myeloid leukemia(AML), breast carcinoma, cholangiocarcinoma, colorectal adenocarcinoma,extrahepatic bile duct adenocarcinoma, female genital tract malignancy,gastric adenocarcinoma, gastroesophageal adenocarcinoma,gastrointestinal stromal tumors (GIST), glioblastoma, head and necksquamous carcinoma, leukemia, liver hepatocellular carcinoma, low gradeglioma, lung bronchioloalveolar carcinoma (BAC), lung non-small celllung cancer (NSCLC), lung small cell cancer (SCLC), lymphoma, malegenital tract malignancy, malignant solitary fibrous tumor of the pleura(MSFT), melanoma, multiple myeloma, neuroendocrine tumor, nodal diffuselarge B-cell lymphoma, non epithelial ovarian cancer (non-EOC), ovariansurface epithelial carcinoma, pancreatic adenocarcinoma, pituitarycarcinomas, oligodendroglioma, prostatic adenocarcinoma, retroperitonealor peritoneal carcinoma, retroperitoneal or peritoneal sarcoma, smallintestinal malignancy, soft tissue tumor, thymic carcinoma, thyroidcarcinoma, or uveal melanoma. The methods of the invention can be usedto characterize these and other cancers. Thus, characterizing aphenotype can be providing a diagnosis, prognosis or theranosis of oneof the cancers disclosed herein.

The phenotype can also be an inflammatory disease, immune disease, orautoimmune disease. For example, the disease may be inflammatory boweldisease (IBD), Crohn's disease (CD), ulcerative colitis (UC), pelvicinflammation, vasculitis, psoriasis, diabetes, autoimmune hepatitis,Multiple Sclerosis, Myasthenia Gravis, Type I diabetes, RheumatoidArthritis, Psoriasis, Systemic Lupus Erythematosis (SLE), Hashimoto'sThyroiditis, Grave's disease, Ankylosing Spondylitis Sjogrens Disease,CREST syndrome, Scleroderma, Rheumatic Disease, organ rejection, PrimarySclerosing Cholangitis, or sepsis.

The phenotype can also comprise a cardiovascular disease, such asatherosclerosis, congestive heart failure, vulnerable plaque, stroke, orischemia. The cardiovascular disease or condition can be high bloodpressure, stenosis, vessel occlusion or a thrombotic event.

The phenotype can also comprise a neurological disease, such as MultipleSclerosis (MS), Parkinson's Disease (PD), Alzheimer's Disease (AD),schizophrenia, bipolar disorder, depression, autism, Prion Disease,Pick's disease, dementia, Huntington disease (HD), Down's syndrome,cerebrovascular disease, Rasmussen's encephalitis, viral meningitis,neurospsychiatric systemic lupus erythematosus (NPSLE), amyotrophiclateral sclerosis, Creutzfeldt-Jacob disease,Gerstmann-Straussler-Scheinker disease, transmissible spongiformencephalopathy, ischemic reperfusion damage (e.g. stroke), brain trauma,microbial infection, or chronic fatigue syndrome. The phenotype may alsobe a condition such as fibromyalgia, chronic neuropathic pain, orperipheral neuropathic pain.

The phenotype may also comprise an infectious disease, such as abacterial, viral or yeast infection. For example, the disease orcondition may be Whipple's Disease, Prion Disease, cirrhosis,methicillin-resistant Staphylococcus aureus, HIV, hepatitis, syphilis,meningitis, malaria, tuberculosis, or influenza. Viral proteins, such asHIV or HCV-like particles can be assessed in a vesicle, to characterizea viral condition.

The phenotype can also comprise a perinatal or pregnancy relatedcondition (e.g. preeclampsia or preterm birth), metabolic disease orcondition, such as a metabolic disease or condition associated with ironmetabolism. For example, hepcidin can be assayed in a vesicle tocharacterize an iron deficiency. The metabolic disease or condition canalso be diabetes, inflammation, or a perinatal condition.

The compositions and methods of the invention can be used tocharacterize these and other diseases and disorders. Thus,characterizing a phenotype can be providing a diagnosis, prognosis ortheranosis of a medical condition, disease or disorder, includingwithout limitation one of the diseases and disorders disclosed herein.

Subject

One or more phenotypes of a subject can be determined by analyzing abiological sample obtained from the subject. A subject or patient caninclude, but is not limited to, mammals such as bovine, avian, canine,equine, feline, ovine, porcine, or primate animals (including humans andnon-human primates). A subject can also include a mammal of importancedue to being endangered, such as a Siberian tiger; or economicimportance, such as an animal raised on a farm for consumption byhumans, or an animal of social importance to humans, such as an animalkept as a pet or in a zoo. Examples of such animals include, but are notlimited to, carnivores such as cats and dogs; swine including pigs, hogsand wild boars; ruminants or ungulates such as cattle, oxen, sheep,giraffes, deer, goats, bison, camels or horses. Also included are birdsthat are endangered or kept in zoos, as well as fowl and moreparticularly domesticated fowl, e.g., poultry, such as turkeys andchickens, ducks, geese, guinea fowl. Also included are domesticatedswine and horses (including race horses). In addition, any animalspecies connected to commercial activities are also included such asthose animals connected to agriculture and aquaculture and otheractivities in which disease monitoring, diagnosis, and therapy selectionare routine practice in husbandry for economic productivity and/orsafety of the food chain.

The subject can have a pre-existing disease or condition, includingwithout limitation cancer. Alternatively, the subject may not have anyknown pre-existing condition. The subject may also be non-responsive toan existing or past treatment, such as a treatment for cancer.

Samples

A sample used and/or assessed via the compositions and methods of theinvention includes any relevant biological sample that can be used tocharacterize a phenotype of interest, including without limitationsections of tissues such as biopsy or tissue removed during surgical orother procedures, bodily fluids, autopsy samples, frozen sections takenfor histological purposes, and cell cultures. Such samples include bloodand blood fractions or products (e.g., serum, buffy coat, plasma,platelets, red blood cells, and the like), sputum, malignant effusion,cheek cells tissue, cultured cells (e.g., primary cultures, explants,and transformed cells), stool, urine, other biological or bodily fluids(e.g., prostatic fluid, gastric fluid, intestinal fluid, renal fluid,lung fluid, cerebrospinal fluid, and the like), etc. The sample cancomprise biological material that is a fresh frozen & formalin fixedparaffin embedded (FFPE) block, formalin-fixed paraffin embedded, or iswithin an RNA preservative+formalin fixative. More than one sample ofmore than one type can be used for each patient.

The sample used in the methods described herein can be a formalin fixedparaffin embedded (FFPE) sample. The FFPE sample can be one or more offixed tissue, unstained slides, bone marrow core or clot, core needlebiopsy, malignant fluids and fine needle aspirate (FNA). In anembodiment, the fixed tissue comprises a tumor containing formalin fixedparaffin embedded (FFPE) block from a surgery or biopsy. In anotherembodiment, the unstained slides comprise unstained, charged, unbakedslides from a paraffin block. In another embodiment, bone marrow core orclot comprises a decalcified core. A formalin fixed core and/or clot canbe paraffin-embedded. In still another embodiment, the core needlebiopsy comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, e.g., 3-6,paraffin embedded biopsy samples. An 18 gauge needle biopsy can be used.The malignant fluid can comprise a sufficient volume of freshpleural/ascitic fluid to produce a 5×5×2 mm cell pellet. The fluid canbe formalin fixed in a paraffin block. In an embodiment, the core needlebiopsy comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, e.g., 4-6,paraffin embedded aspirates.

A sample may be processed according to techniques understood by those inthe art. A sample can be without limitation fresh, frozen or fixed cellsor tissue. In some embodiments, a sample comprises formalin-fixedparaffin-embedded (FFPE) tissue, fresh tissue or fresh frozen (FF)tissue. A sample can comprise cultured cells, including primary orimmortalized cell lines derived from a subject sample. A sample can alsorefer to an extract from a sample from a subject. For example, a samplecan comprise DNA, RNA or protein extracted from a tissue or a bodilyfluid. Many techniques and commercial kits are available for suchpurposes. The fresh sample from the individual can be treated with anagent to preserve RNA prior to further processing, e.g., cell lysis andextraction. Samples can include frozen samples collected for otherpurposes. Samples can be associated with relevant information such asage, gender, and clinical symptoms present in the subject; source of thesample; and methods of collection and storage of the sample. A sample istypically obtained from a subject, e.g., a human subject.

A biopsy comprises the process of removing a tissue sample fordiagnostic or prognostic evaluation, and to the tissue specimen itself.Any biopsy technique known in the art can be applied to the molecularprofiling methods of the present invention. The biopsy technique appliedcan depend on the tissue type to be evaluated (e.g., colon, prostate,kidney, bladder, lymph node, liver, bone marrow, blood cell, lung,breast, etc.), the size and type of the tumor (e.g., solid or suspended,blood or ascites), among other factors. Representative biopsy techniquesinclude, but are not limited to, excisional biopsy, incisional biopsy,needle biopsy, surgical biopsy, and bone marrow biopsy. An “excisionalbiopsy” refers to the removal of an entire tumor mass with a smallmargin of normal tissue surrounding it. An “incisional biopsy” refers tothe removal of a wedge of tissue that includes a cross-sectionaldiameter of the tumor. The invention can make use a “core-needle biopsy”of the tumor mass, or a “fine-needle aspiration biopsy” which generallyobtains a suspension of cells from within the tumor mass. Biopsytechniques are discussed, for example, in Harrison's Principles ofInternal Medicine, Kasper, et al., eds., 16th ed., 2005, Chapter 70, andthroughout Part V.

Standard molecular biology techniques known in the art and notspecifically described are generally followed as in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York (1989), and as in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and as inPerbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, NewYork (1988), and as in Watson et al., Recombinant DNA, ScientificAmerican Books, New York and in Birren et al (eds) Genome Analysis: ALaboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press,New York (1998) and methodology as set forth in U.S. Pat. Nos.4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 andincorporated herein by reference. Polymerase chain reaction (PCR) can becarried out generally as in PCR Protocols: A Guide to Methods andApplications, Academic Press, San Diego, Calif. (1990).

The biological sample assessed using the compositions and methods of theinvention can be any useful bodily or biological fluid, including butnot limited to peripheral blood, sera, plasma, ascites, urine,cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid,aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolarlavage fluid, semen (including prostatic fluid), Cowper's fluid orpre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair,tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid,lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum,vomit, vaginal secretions, mucosal secretion, stool water, pancreaticjuice, lavage fluids from sinus cavities, bronchopulmonary aspirates orother lavage fluids, cells, cell culture, or a cell culture supernatant.A biological sample may also include the blastocyl cavity, umbilicalcord blood, or maternal circulation which may be of fetal or maternalorigin. The biological sample may also be a cell culture, tissue sampleor biopsy from which microvesicles, circulating tumor cells (CTCs), andother circulating biomarkers may be obtained. For example, cells ofinterest can be cultured and microvesicles isolated from the culture. Invarious embodiments, biomarkers or more particularly biosignaturesdisclosed herein can be assessed directly from such biological samples(e.g., identification of presence or levels of nucleic acid orpolypeptide biomarkers or functional fragments thereof) using variousmethods, such as extraction of nucleic acid molecules from blood,plasma, serum or any of the foregoing biological samples, use of proteinor antibody arrays to identify polypeptide (or functional fragment)biomarker(s), as well as other array, sequencing, PCR and proteomictechniques known in the art for identification and assessment of nucleicacid and polypeptide molecules. In addition, one or more componentspresent in such samples can be first isolated or enriched and furtherprocessed to assess the presence or levels of selected biomarkers, toassess a given biosignature (e.g., isolated microvesicles prior toprofiling for protein and/or nucleic acid biomarkers).

Table 1 presents a non-limiting listing of diseases, conditions, orbiological states and corresponding biological samples that may be usedfor analysis according to the methods of the invention.

TABLE 1 Examples of Biological Samples for Various Diseases, Conditions,or Biological States Illustrative Disease, Condition or Biological StateIllustrative Biological Samples Cancers/neoplasms affecting thefollowing tissue Tumor, blood, serum, plasma, cerebrospinal fluidtypes/bodily systems: breast, lung, ovarian, colon, (CSF), urine,sputum, ascites, synovial fluid, rectal, prostate, pancreatic, brain,bone, connective semen, nipple aspirates, saliva, bronchoalveolartissue, glands, skin, lymph, nervous system, lavage fluid, tears,oropharyngeal washes, feces, endocrine, germ cell, genitourinary,peritoneal fluids, pleural effusion, sweat, tears, hematologic/blood,bone marrow, muscle, eye, aqueous humor, pericardial fluid, lymph,chyme, esophageal, fat tissue, thyroid, pituitary, spinal chyle, bile,stool water, amniotic fluid, breast milk, cord, bile duct, heart, gallbladder, bladder, testes, pancreatic juice, cerumen, Cowper's fluid orpre- cervical, endometrial, renal, ovarian, ejaculatory fluid, femaleejaculate, interstitial fluid, digestive/gastrointestinal, stomach, headand neck, menses, mucus, pus, sebum, vaginal lubrication, liver,leukemia, respiratory/thorasic, cancers of vomit unknown primary (CUP)Neurodegenerative/neurological disorders: Blood, serum, plasma, CSF,urine Parkinson's disease, Alzheimer's Disease and multiple sclerosis,Schizophrenia, and bipolar disorder, spasticity disorders, epilepsyCardiovascular Disease: atherosclerosis, Blood, serum, plasma, CSF,urine cardiomyopathy, endocarditis, vunerable plaques, infection Stroke:ischemic, intracerebral hemorrhage, Blood, serum, plasma, CSF, urinesubarachnoid hemorrhage, transient ischemic attacks (TIA) Paindisorders: peripheral neuropathic pain and Blood, serum, plasma, CSF,urine chronic neuropathic pain, and fibromyalgia, Autoimmune disease:systemic and localized Blood, serum, plasma, CSF, urine, synovial fluiddiseases, rheumatic disease, Lupus, Sjogren's syndrome Digestive systemabnormalities: Barrett's Blood, serum, plasma, CSF, urine esophagus,irritable bowel syndrome, ulcerative colitis, Crohn's disease,Diverticulosis and Diverticulitis, Celiac Disease Endocrine disorders:diabetes mellitus, various Blood, serum, plasma, CSF, urine forms ofThyroiditis, adrenal disorders, pituitary disorders Diseases anddisorders of the skin: psoriasis Blood, serum, plasma, CSF, urine,synovial fluid, tears Urological disorders: benign prostatic hypertrophyBlood, serum, plasma, urine (BPH), polycystic kidney disease,interstitial cystitis Hepatic disease/injury: Cirrhosis, induced Blood,serum, plasma, urine hepatotoxicity (due to exposure to natural orsynthetic chemical sources) Kidney disease/injury: acute, sub-acute,chronic Blood, serum, plasma, urine conditions, Podocyte injury, focalsegmental glomerulosclerosis Endometriosis Blood, serum, plasma, urine,vaginal fluids Osteoporosis Blood, serum, plasma, urine, synovial fluidPancreatitis Blood, serum, plasma, urine, pancreatic juice Asthma Blood,serum, plasma, urine, sputum, bronchiolar lavage fluid Allergies Blood,serum, plasma, urine, sputum, bronchiolar lavage fluid Prion-relateddiseases Blood, serum, plasma, CSF, urine Viral Infections: HIV/AIDSBlood, serum, plasma, urine Sepsis Blood, serum, plasma, urine, tears,nasal lavage Organ rejection/transplantation Blood, serum, plasma,urine, various lavage fluids Differentiating conditions: adenoma versusBlood, serum, plasma, urine, sputum, feces, colonic hyperplastic polyp,irritable bowel syndrome (IBS) lavage fluid versus normal, classifyingDukes stages A, B, C, and/or D of colon cancer, adenoma with low-gradehyperplasia versus high-grade hyperplasia, adenoma versus normal,colorectal cancer versus normal, IBS versus. ulcerative colitis (UC)versus Crohn's disease (CD), Pregnancy related physiological states,conditions, or Maternal serum, plasma, amniotic fluid, cord bloodaffiliated diseases: genetic risk, adverse pregnancy outcomes

The methods of the invention can be used to characterize a phenotypeusing a blood sample or blood derivative. Blood derivatives includeplasma and serum. Blood plasma is the liquid component of whole blood,and makes up approximately 55% of the total blood volume. It is composedprimarily of water with small amounts of minerals, salts, ions,nutrients, and proteins in solution. In whole blood, red blood cells,leukocytes, and platelets are suspended within the plasma. Blood serumrefers to blood plasma without fibrinogen or other clotting factors(i.e., whole blood minus both the cells and the clotting factors).

The biological sample may be obtained through a third party, such as aparty not performing the analysis of the sample. For example, the samplemay be obtained through a clinician, physician, or other health caremanager of a subject from which the sample is derived. Alternatively,the biological sample may obtained by the same party analyzing thesample. In addition, biological samples be assayed, are archived (e.g.,frozen) or otherwise stored in under preservative conditions.

In various embodiments, the biological sample comprises a microvesicleor cell membrane fragment that is derived from a cell of origin andavailable extracellularly in a subject's biological fluid orextracellular milieu. Methods of the invention may include assessing oneor more such microvesicles, including assessing populations thereof. Avesicle or microvesicle, as used herein, is a membrane vesicle that isshed from cells. Vesicles or membrane vesicles include withoutlimitation: circulating microvesicles (cMVs), microvesicle, exosome,nanovesicle, dexosome, bleb, blebby, prostasome, microparticle,intralumenal vesicle, membrane fragment, intralumenal endosomal vesicle,endosomal-like vesicle, exocytosis vehicle, endosome vesicle, endosomalvesicle, apoptotic body, multivesicular body, secretory vesicle,phospholipid vesicle, liposomal vesicle, argosome, texasome, secresome,tolerosome, melanosome, oncosome, or exocytosed vehicle. Furthermore,although vesicles may be produced by different cellular processes, themethods of the invention are not limited to or reliant on any onemechanism, insofar as such vesicles are present in a biological sampleand are capable of being characterized by the methods disclosed herein.Unless otherwise specified, methods that make use of a species ofvesicle can be applied to other types of vesicles. Vesicles comprisespherical structures with a lipid bilayer similar to cell membraneswhich surrounds an inner compartment which can contain solublecomponents, sometimes referred to as the payload. In some embodiments,the methods of the invention make use of exosomes, which are smallsecreted vesicles of about 40-100 nm in diameter. For a review ofmembrane vesicles, including types and characterizations, see Thery etal., Nat Rev Immunol. 2009 August; 9(8):581-93. Some properties ofdifferent types of vesicles include those in Table 2:

TABLE 2 Vesical Properties Feature Exosomes Microvesicles EctosomesMembrane particles Exosome-like vesicles Apoptotic vesicles Size 50-100nm 100-1,000 nm 50-200 nm 50-80 nm 20-50 nm 50-500 nm Density in1.13-1.19 g/ml 1.04-1.07 g/ml 1.1 g/ml 1.16-1.28 g/ml sucrose EM Cupshape Irregular Bilamellar Round Irregular Heterogeneous appearanceshape, round shape electron structures dense Sedimentation 100,000 g10,000 g 160,000-200,000 g 100,000-200,000 g 175,000 g 1,200 g, 10,000g, 100,000 g Lipid Enriched in Expose PPS Enriched in No lipidcomposition cholesterol, cholesterol rafts sphingomyelin and andceramide; diacylglycerol; contains lipid expose PPS rafts; expose PPSMajor protein Tetraspanins Integrins, CR1 and CD133; no TNFRI Histonesmarkers (e.g., CD63, selectins and proteolytic CD63 CD9), Alix, CD40ligand enzymes; no TSG101 CD63 Intracellular Internal Plasma PlasmaPlasma origin compartments membrane membrane membrane (endosomes)Abbreviations: phosphatidylserine (PPS); electron microscopy (EM)

Vesicles include shed membrane bound particles, or “microparticles,”that are derived from either the plasma membrane or an internalmembrane. Vesicles can be released into the extracellular environmentfrom cells. Cells releasing vesicles include without limitation cellsthat originate from, or are derived from, the ectoderm, endoderm, ormesoderm. The cells may have undergone genetic, environmental, and/orany other variations or alterations. For example, the cell can be tumorcells. A vesicle can reflect any changes in the source cell, and therebyreflect changes in the originating cells, e.g., cells having variousgenetic mutations. In one mechanism, a vesicle is generatedintracellularly when a segment of the cell membrane spontaneouslyinvaginates and is ultimately exocytosed (see for example, Keller etal., Immunol. Lett. 107 (2): 102-8 (2006)). Vesicles also includecell-derived structures bounded by a lipid bilayer membrane arising fromboth herniated evagination (blebbing) separation and sealing of portionsof the plasma membrane or from the export of any intracellularmembrane-bounded vesicular structure containing variousmembrane-associated proteins of tumor origin, including surface-boundmolecules derived from the host circulation that bind selectively to thetumor-derived proteins together with molecules contained in the vesiclelumen, including but not limited to tumor-derived microRNAs orintracellular proteins. Blebs and blebbing are further described inCharras et al., Nature Reviews Molecular and Cell Biology, Vol. 9, No.11, p. 730-736 (2008). A vesicle shed into circulation or bodily fluidsfrom tumor cells may be referred to as a “circulating tumor-derivedvesicle.” When such vesicle is an exosome, it may be referred to as acirculating-tumor derived exosome (CTE). In some instances, a vesiclecan be derived from a specific cell of origin. CTE, as with acell-of-origin specific vesicle, typically have one or more uniquebiomarkers that permit isolation of the CTE or cell-of-origin specificvesicle, e.g., from a bodily fluid and sometimes in a specific manner.For example, a cell or tissue specific markers are used to identify thecell of origin. Examples of such cell or tissue specific markers aredisclosed herein and can further be accessed in the Tissue-specific GeneExpression and Regulation (TiGER) Database, available atbioinfo.wilmer.jhu.edu/tiger/; Liu et al. (2008) TiGER: a database fortissue-specific gene expression and regulation. BMC Bioinformatics.9:271; TissueDistributionDBs, available atgenome.dkfz-heidelberg.de/menu/tissue_db/index.html.

A vesicle can have a diameter of greater than about 10 nm, 20 nm, or 30nm. A vesicle can have a diameter of greater than 40 nm, 50 nm, 100 nm,200 nm, 500 nm, 1000 nm, 1500 nm, 2000 nm or greater than 10,000 nm. Avesicle can have a diameter of about 20-2000 nm, about 20-1500 nm, about30-1000 nm, about 30-800 nm, about 30-200 nm, or about 30-100 nm. Insome embodiments, the vesicle has a diameter of less than 10,000 nm,2000 nm, 1500 nm, 1000 nm, 800 nm, 500 nm, 200 nm, 100 nm, 50 nm, 40 nm,30 nm, 20 nm or less than 10 nm. As used herein the term “about” inreference to a numerical value means that variations of 10% above orbelow the numerical value are within the range ascribed to the specifiedvalue. Typical sizes for various types of vesicles are shown in Table 2.Vesicles can be assessed to measure the diameter of a single vesicle orany number of vesicles. For example, the range of diameters of a vesiclepopulation or an average diameter of a vesicle population can bedetermined. Vesicle diameter can be assessed using methods known in theart, e.g., imaging technologies such as electron microscopy. In anembodiment, a diameter of one or more vesicles is determined usingoptical particle detection. See, e.g., U.S. Pat. No. 7,751,053, entitled“Optical Detection and Analysis of Particles” and issued Jul. 6, 2010;and U.S. Pat. No. 7,399,600, entitled “Optical Detection and Analysis ofParticles” and issued Jul. 15, 2010.

In some embodiments, the methods of the invention comprise assessingvesicles directly such as in a biological sample without priorisolation, purification, or concentration from the biological sample.For example, the amount of vesicles in the sample can by itself providea biosignature that provides a diagnostic, prognostic or theranosticdetermination. Alternatively, the vesicle in the sample may be isolated,captured, purified, or concentrated from a sample prior to analysis. Asnoted, isolation, capture or purification as used herein comprisespartial isolation, partial capture or partial purification apart fromother components in the sample. Vesicle isolation can be performed usingvarious techniques as described herein, e.g., chromatography,filtration, centrifugation, flow cytometry, affinity capture (e.g., to aplanar surface or bead), and/or using microfluidics. FIGS. 10B-C presentan overview of a method of the invention for assessing microvesiclesusing an aptamer pool.

Vesicles such as exosomes can be assessed to provide a phenotypiccharacterization by comparing vesicle characteristics to a reference. Insome embodiments, surface antigens on a vesicle are assessed. Thesurface antigens can provide an indication of the anatomical originand/or cellular of the vesicles and other phenotypic information, e.g.,tumor status. For example, wherein vesicles found in a patient sample,e.g., a bodily fluid such as blood, serum or plasma, are assessed forsurface antigens indicative of colorectal origin and the presence ofcancer. The surface antigens may comprise any informative biologicalentity that can be detected on the vesicle membrane surface, includingwithout limitation surface proteins, lipids, carbohydrates, and othermembrane components. For example, positive detection of colon derivedvesicles expressing tumor antigens can indicate that the patient hascolorectal cancer. As such, methods of the invention can be used tocharacterize any disease or condition associated with an anatomical orcellular origin, by assessing, for example, disease-specific andcell-specific biomarkers of one or more vesicles obtained from asubject.

In another embodiment, the methods of the invention comprise assessingone or more vesicle payload to provide a phenotypic characterization.The payload with a vesicle comprises any informative biological entitythat can be detected as encapsulated within the vesicle, includingwithout limitation proteins and nucleic acids, e.g., genomic or cDNA,mRNA, or functional fragments thereof, as well as microRNAs (miRs). Inaddition, methods of the invention are directed to detecting vesiclesurface antigens (in addition or exclusive to vesicle payload) toprovide a phenotypic characterization. For example, vesicles can becharacterized by using binding agents (e.g., antibodies or aptamers)that are specific to vesicle surface antigens, and the bound vesiclescan be further assessed to identify one or more payload componentsdisclosed therein. As described herein, the levels of vesicles withsurface antigens of interest or with payload of interest can be comparedto a reference to characterize a phenotype. For example, overexpressionin a sample of cancer-related surface antigens or vesicle payload, e.g.,a tumor associated mRNA or microRNA, as compared to a reference, canindicate the presence of cancer in the sample. The biomarkers assessedcan be present or absent, increased or reduced based on the selection ofthe desired target sample and comparison of the target sample to thedesired reference sample. Non-limiting examples of target samplesinclude: disease; treated/not-treated; different time points, such as ain a longitudinal study; and non-limiting examples of reference sample:non-disease; normal; different time points; and sensitive or resistantto candidate treatment(s).

Diagnostic Methods

The aptamers of the invention can be used in various methods to assesspresence or level of biomarkers in a biological sample, e.g., biologicalentities of interest such as proteins, nucleic acids, or microvesicles.The biological entities can be part of larger entities, such ascomplexes, cells or tissue, or can be circulating in bodily fluids. Theaptamers may be used to assess presence or level of the targetmolecule/s. Therefore, in various embodiments of the invention directedto diagnostics, prognostics or theranostics, one or more aptamers of theinvention are configured in a ligand-target based assay, where one ormore aptamer of the invention is contacted with a selected biologicalsample, where the or more aptamer associates with or binds to its targetmolecules. Aptamers of the invention are used to identify candidatebiosignatures based on the biological samples assessed and biomarkersdetected. In some embodiments, aptamer or oligonucleotide probes, orlibraries thereof, may themselves provide a biosignature for aparticular condition or disease. A biosignature refers to a biomarkerprofile of a biological sample comprising a presence, level or othercharacteristic that can be assessed (including without limitation asequence, mutation, rearrangement, translocation, deletion, epigeneticmodification, methylation, post-translational modification, allele,activity, complex partners, stability, half life, and the like) of oneor more biomarker of interest. Biosignatures can be used to evaluatediagnostic and/or prognostic criteria such as presence of disease,disease staging, disease monitoring, disease stratification, orsurveillance for detection, metastasis or recurrence or progression ofdisease. For example, methods of the invention using aptamers againstmicrovesicle surface antigen are useful for correlating a biosignaturecomprising microvesicle antigens to a selected condition or disease. Asanother example, methods of the invention using aptamers against tissueare useful for correlating a biosignature comprising tissue antigens toa selected condition or disease. A biosignature can also be usedclinically in making decisions concerning treatment modalities includingtherapeutic intervention. A biosignature can further be used clinicallyto make treatment decisions, including whether to perform surgery orwhat treatment standards should be used along with surgery (e.g., eitherpre-surgery or post-surgery). As an illustrative example, a biosignatureof circulating biomarkers or biomarkers displayed on fixed tissue mayindicate an aggressive form of cancer and may call for a more aggressivesurgical procedure and/or more aggressive therapeutic regimen to treatthe patient.

Characterizing a phenotype, such as providing a diagnosis, prognosis ortheranosis, may comprise comparing a biosignature to a reference. Forexample, the level of a biomarker in a diseased state may be elevated orreduced as compared to a reference control without the disease, or witha different state of the disease. An oligonucleotide probe libraryaccording to the invention may be engineered to detect a certainphenotype and not another phenotype. As a non-limiting example, theoligonucleotide probe library may stain a cancer tissue using animmunoassay but not a non-cancer reference tissue. Alternately, theoligonucleotide probe library may stain a cancer tissue using animmunoassay at a detectable higher level than a non-cancer referencetissue. One of skill will appreciate that one may engineer anoligonucleotide probe library to stain a non-cancer tissue using animmunoassay at a detectable higher level than cancer tissue as well.

A biosignature can be used in any methods disclosed herein, e.g., toassess whether a subject is afflicted with disease, is at risk fordeveloping disease or to assess the stage or progression of the disease.For example, a biosignature can be used to assess whether a subject hasprostate cancer, colon cancer, or other cancer as described herein. See,e.g., section labeled “Phenotypes.” Furthermore, a biosignature can beused to determine a stage of a disease or condition, such as cancer.

A biosignature/biomarker profile comprising a microvesicle can includeassessment of payload within the microvesicle. For example, one or moreaptamer of the invention can be used to capture a microvesiclepopulation, thereby providing readout of microvesicle antigens, and thenthe payload content within the captured microvesicles can be assessed,thereby providing further biomarker readout of the payload content.

A biosignature for characterizing a phenotype may comprise any number ofuseful criteria. The term “phenotype” as used herein can mean any traitor characteristic that is attributed to a biosignature/biomarkerprofile. A phenotype can be detected or identified in part or in wholeusing the compositions and/or methods of the invention. In someembodiments, at least one criterion is used for each biomarker. In someembodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50,60, 70, 80, 90 or at least 100 criteria are used. For example, for thecharacterizing of a cancer, a number of different criteria can be usedwhen the subject is diagnosed with a cancer: 1) if the amount of abiomarker in a sample from a subject is higher than a reference value;2) if the amount of a biomarker within specific cell types or specificmicrovesicles (e.g., microvesicles derived from a specific tissue ororgan) is higher than a reference value; or 3) if the amount of abiomarker within a cell, tissue or microvesicle with one or more cancerspecific biomarkers is higher than a reference value Similar rules canapply if the amount of the biomarkers is less than or the same as thereference. The method can further include a quality control measure,such that the results are provided for the subject if the samples meetthe quality control measure. In some embodiments, if the criteria aremet but the quality control is questionable, the subject is reassessed.

A biosignature can be used in therapy related diagnostics to providetests useful to diagnose a disease or choose the correct treatmentregimen, such as provide a theranosis. Theranostics includes diagnostictesting that provides the ability to affect therapy or treatment of adiseased state. Theranostics testing provides a theranosis in a similarmanner that diagnostics or prognostic testing provides a diagnosis orprognosis, respectively. As used herein, theranostics encompasses anydesired form of therapy related testing, including predictive medicine,personalized medicine, integrated medicine, pharmacodiagnostics andDx/Rx partnering. Therapy related tests can be used to predict andassess drug response in individual subjects, i.e., to providepersonalized medicine. Predicting a drug response can be determiningwhether a subject is a likely responder or a likely non-responder to acandidate therapeutic agent, e.g., before the subject has been exposedor otherwise treated with the treatment. Assessing a drug response canbe monitoring a response to a drug, e.g., monitoring the subject'simprovement or lack thereof over a time course after initiating thetreatment. Therapy related tests are useful to select a subject fortreatment who is particularly likely to benefit from the treatment or toprovide an early and objective indication of treatment efficacy in anindividual subject. Thus, a biosignature as disclosed herein mayindicate that treatment should be altered to select a more promisingtreatment, thereby avoiding the great expense of delaying beneficialtreatment and avoiding the financial and morbidity costs ofadministering an ineffective drug(s).

The compositions and methods of the invention can be used to identify ordetect a biosignature associated with a variety of diseases anddisorders, which include, but are not limited to cardiovascular disease,cancer, infectious diseases, sepsis, neurological diseases, centralnervous system related diseases, endovascular related diseases, andautoimmune related diseases. Therapy related diagnostics also aid in theprediction of drug toxicity, drug resistance or drug response. Therapyrelated tests may be developed in any suitable diagnostic testingformat, which include, but are not limited to, e.g., immunohistochemicaltests, clinical chemistry, immunoassay, cell-based technologies, nucleicacid tests or body imaging methods. Therapy related tests can furtherinclude but are not limited to, testing that aids in the determinationof therapy, testing that monitors for therapeutic toxicity, or responseto therapy testing. Thus, a biosignature can be used to predict ormonitor a subject's response to a treatment. A biosignature can bedetermined at different time points for a subject after initiating,removing, or altering a particular treatment.

In some embodiments, the compositions and methods of the inventionprovide for a determination or prediction as to whether a subject isresponding to a treatment is made based on a change in the amount of oneor more components of a biosignature (e.g., biomarkers of interest), anamount of one or more components of a particular biosignature, or thebiosignature detected for the components. In another embodiment, asubject's condition is monitored by determining a biosignature atdifferent time points. The progression, regression, or recurrence of acondition is determined. Response to therapy can also be measured over atime course. Thus, the invention provides a method of monitoring astatus of a disease or other medical condition in a subject, comprisingisolating or detecting a biosignature from a biological sample from thesubject, detecting the overall amount of the components of a particularbiosignature, or detecting the biosignature of one or more components(such as the presence, absence, or expression level of a biomarker). Thebiosignatures are used to monitor the status of the disease orcondition.

One or more novel biosignatures can also be identified by the methods ofthe invention. For example, one or more vesicles can be isolated from asubject that responds to a drug treatment or treatment regimen andcompared to a reference, such as another subject that does not respondto the drug treatment or treatment regimen. Differences between thebiosignatures can be determined and used to identify other subjects asresponders or non-responders to a particular drug or treatment regimen.

In some embodiments, a biosignature is used to determine whether aparticular disease or condition is resistant to a drug, in which case aphysician need not waste valuable time with such drug treatment. Toobtain early validation of a drug choice or treatment regimen, abiosignature is determined for a sample obtained from a subject. Thebiosignature is used to assess whether the particular subject's diseasehas the biomarker associated with drug resistance. Such a determinationenables doctors to devote critical time as well as the patient'sfinancial resources to effective treatments.

Biosignatures can be used in the theranosis of diseases such as cancer,e.g., identifying whether a subject suffering from a disease is a likelyresponder or non-responder to a particular treatment. The subjectmethods can be used to theranose cancers including without limitationthose listed herein, e.g., in the “Phenotypes” section herein. Theseinclude without limitation lung cancer, non-small cell lung cancer smallcell lung cancer (including small cell carcinoma (oat cell cancer),mixed small cell/large cell carcinoma, and combined small cellcarcinoma), colon cancer, breast cancer, prostate cancer, liver cancer,pancreatic cancer, brain cancer, kidney cancer, ovarian cancer, stomachcancer, melanoma, bone cancer, gastric cancer, breast cancer, glioma,glioblastoma, hepatocellular carcinoma, papillary renal carcinoma, headand neck squamous cell carcinoma, leukemia, lymphoma, myeloma, or othersolid tumors.

A biosignature of circulating biomarkers, including markers associatedwith a component present in a biological sample (e.g., cell,cell-fragment, cell-derived microvesicle), in a sample from a subjectsuffering from a cancer can be used select a candidate treatment for thesubject. The biosignature can be determined according to the methods ofthe invention presented herein. In some embodiments, the candidatetreatment comprises a standard of care for the cancer. The treatment canbe a cancer treatment such as radiation, surgery, chemotherapy or acombination thereof. The cancer treatment can be a therapeutic such asanti-cancer agents and chemotherapeutic regimens. Further drugassociations and rules that are used in embodiments of the invention arefound in PCT/US2007/69286, filed May 18, 2007; PCT/US2009/60630, filedOct. 14, 2009; PCT/2010/000407, filed Feb. 11, 2010; PCT/US12/41393,filed Jun. 7, 2012; PCT/US2013/073184, filed Dec. 4, 2013;PCT/US2010/54366, filed Oct. 27, 2010; PCT/US11/67527, filed Dec. 28,2011; PCT/US15/13618, filed Jan. 29, 2015; and PCT/US16/20657, filedMar. 3, 2016; each of which applications is incorporated herein byreference in its entirety.

Biomarkers

The methods and compositions of the invention can be used in assays todetect the presence or level of one or more biomarker of interest. Giventhe adaptable nature of the invention, the biomarker can be any usefulbiomarker including those disclosed herein or in the literature, or tobe discovered. In an embodiment, the biomarker comprises a protein orpolypeptide. As used herein, “protein,” “polypeptide” and “peptide” areused interchangeably unless stated otherwise. The biomarker can be anucleic acid, including DNA, RNA, and various subspecies of any thereofas disclosed herein or known in the art. The biomarker can comprise alipid. The biomarker can comprise a carbohydrate. The biomarker can alsobe a complex, e.g., a complex comprising protein, nucleic acids, lipidsand/or carbohydrates. In some embodiments, the biomarker comprises amicrovesicle. In an embodiment, the invention provides a method whereina pool of aptamers is used to assess the presence and/or level of apopulation of microvesicles of interest without knowing the precisemicrovesicle antigen targeted by each member of the pool. See, e.g.,FIGS. 10B-C. In other cases, biomarkers associated with microvesiclesare assessed according to the methods of the invention. See, e.g., FIG.10A. The oligonucleotide pools of the invention can also used to assesscells and tissue whether or not the target biomarkers of the individualoligonucleotide aptamers are known. The invention further includesdetermining the targets of such oligonucleotide aptamer pools andmembers thereof. See Examples 19-31.

A biosignature may comprise one type of biomarker or multiple types ofbiomarkers. As a non-limiting example, a biosignature can comprisemultiple proteins, multiple nucleic acids, multiple lipids, multiplecarbohydrates, multiple biomarker complexes, multiple microvesicles, ora combination of any thereof. For example, the biosignature may compriseone or more microvesicle, one or more protein, and one or more microRNA,wherein the one or more protein and/or one or more microRNA isoptionally in association with the microvesicle as a surface antigenand/or payload, as appropriate. As another example, the biosignature maybe an oligonucleotide pool signature, and the members of theoligonucleotide pool can associate with various biomarker or multipletypes of biomarkers.

In some embodiments, microvesicles are detected using vesicle surfaceantigens. A commonly expressed vesicle surface antigen can be referredto as a “housekeeping protein,” or general vesicle biomarker. Thebiomarker can be CD63, CD9, CD81, CD82, CD37, CD53, Rab-5b, Annexin V orMFG-E8. Tetraspanins, a family of membrane proteins with fourtransmembrane domains, can be used as general vesicle biomarkers. Thetetraspanins include CD151, CD53, CD37, CD82, CD81, CD9 and CD63. Therehave been over 30 tetraspanins identified in mammals, including theTSPAN1 (TSP-1), TSPAN2 (TSP-2), TSPAN3 (TSP-3), TSPAN4 (TSP-4, NAG-2),TSPAN5 (TSP-5), TSPAN6 (TSP-6), TSPAN7 (CD231, TALLA-1, A15), TSPAN8(CO-029), TSPAN9 (NET-5), TSPAN10 (Oculospanin), TSPAN11 (CD151-like),TSPAN12 (NET-2), TSPAN13 (NET-6), TSPAN14, TSPAN15 (NET-7), TSPAN16(TM4-B), TSPAN17, TSPAN18, TSPAN19, TSPAN20 (UP1b, UPK1B), TSPAN21(UP1a, UPK1A), TSPAN22 (RDS, PRPH2), TSPAN23 (ROM1), TSPAN24 (CD151),TSPAN25 (CD53), TSPAN26 (CD37), TSPAN27 (CD82), TSPAN28 (CD81), TSPAN29(CD9), TSPAN30 (CD63), TSPAN31 (SAS), TSPAN32 (TSSC6), TSPAN33, andTSPAN34. Other commonly observed vesicle markers include those listed inTable 3. One or more of these proteins can be useful biomarkers for thecharacterizing a phenotype using the subject methods and compositions.

TABLE 3 Proteins Observed in Microvesicles from Multiple Cell TypesClass Protein Antigen Presentation MHC class I, MHC class II, Integrins,Alpha 4 beta 1, Alpha M beta 2, Beta 2 Immunoglobulin family ICAM1/CD54,P-selection Cell-surface peptidases Dipeptidylpeptidase IV/CD26,Aminopeptidase n/CD13 Tetraspanins CD151, CD53, CD37, CD82, CD81, CD9and CD63 Heat-shock proteins Hsp70, Hsp84/90 Cytoskeletal proteinsActin, Actin-binding proteins, Tubulin Membrane transport Annexin I,Annexin II, Annexin IV, Annexin V, Annexin VI, and fusionRAB7/RAP1B/RADGDI Signal transduction Gi2alpha/14-3-3, CBL/LCK Abundantmembrane CD63, GAPDH, CD9, CD81, ANXA2, ENO1, SDCBP, MSN, MFGE8,proteins EZR, GK, ANXA1, LAMP2, DPP4, TSG101, HSPA1A, GDI2, CLTC, LAMP1,Cd86, ANPEP, TFRC, SLC3A2, RDX, RAP1B, RAB5C, RAB5B, MYH9, ICAM1, FN1,RAB11B, PIGR, LGALS3, ITGB1, EHD1, CLIC1, ATP1A1, ARF1, RAP1A, P4HB,MUC1, KRT10, HLA- A, FLOT1, CD59, C1orf58, BASP1, TACSTD1, STOM OtherTransmembrane Cadherins: CDH1, CDH2, CDH12, CDH3, Deomoglein, DSG1,DSG2, Proteins DSG3, DSG4, Desmocollin, DSC1, DSC2, DSC3,Protocadherins, PCDH1, PCDH10, PCDH11x, PCDH11y, PCDH12, FAT, FAT2,FAT4, PCDH15, PCDH17, PCDH18, PCDH19; PCDH20; PCDH7, PCDH8, PCDH9,PCDHA1, PCDHA10, PCDHA11, PCDHA12, PCDHA13, PCDHA2, PCDHA3, PCDHA4,PCDHA5, PCDHA6, PCDHA7, PCDHA8, PCDHA9, PCDHAC1, PCDHAC2, PCDHB1,PCDHB10, PCDHB11, PCDHB12, PCDHB13, PCDHB14, PCDHB15, PCDHB16, PCDHB17,PCDHB18, PCDHB2, PCDHB3, PCDHB4, PCDHB5, PCDHB6, PCDHB7, PCDHB8, PCDHB9,PCDHGA1, PCDHGA10, PCDHGA11, PCDHGA12, PCDHGA2; PCDHGA3, PCDHGA4,PCDHGA5, PCDHGA6, PCDHGA7, PCDHGA8, PCDHGA9, PCDHGB1, PCDHGB2, PCDHGB3,PCDHGB4, PCDHGB5, PCDHGB6, PCDHGB7, PCDHGC3, PCDHGC4, PCDHGC5, CDH9(cadherin 9, type 2 (T1-cadherin)), CDH10 (cadherin 10, type 2 (T2-cadherin)), CDH5 (VE-cadherin (vascular endothelial)), CDH6 (K- cadherin(kidney)), CDH7 (cadherin 7, type 2), CDH8 (cadherin 8, type 2), CDH11(OB-cadherin (osteoblast)), CDH13 (T-cadherin - H-cadherin (heart)),CDH15 (M-cadherin (myotubule)), CDH16 (KSP-cadherin), CDH17 (LI cadherin(liver-intestine)), CDH18 (cadherin 18, type 2), CDH19 (cadherin 19,type 2), CDH20 (cadherin 20, type 2), CDH23 (cadherin 23, (neurosensoryepithelium)), CDH10, CDH11, CDH13, CDH15, CDH16, CDH17, CDH18, CDH19,CDH22, CDH23, CDH24, CDH26, CDH28, CDH4, CDH5, CDH6, CDH7, CDH8, CDH9,CELSR1, CELSR2, CELSR3, CLSTN1, CLSTN2, CLSTN3, DCHS1, DCHS2, LOC389118,PCLKC, RESDA1, RET

Any of the types of biomarkers described herein can be used and/orassessed via the subject methods and compositions. Exemplary biomarkersinclude without limitation those in Table 4. The markers can be detectedas protein, RNA or DNA as appropriate, which can be circulating freelyor in a complex with other biological molecules. As desired, the markersin Table 4 can also be used to detect tumor tissue or for capture and/ordetection of vesicles for characterizing phenotypes as disclosed herein.In some cases, multiple capture and/or detectors are used to enhance thecharacterization. The markers can be detected as vesicle surfaceantigens and/or vesicle payload. The “Illustrative Class” indicatesindications for which the markers are known markers. Those of skill willappreciate that the markers can also be used in alternate settings incertain instances. For example, a marker which can be used tocharacterize one type of disease may also be used to characterizeanother disease as appropriate. Consider a non-limiting example of atumor marker which can be used as a biomarker for tumors from variouslineages. The biomarker references in Tables 3 and 4, or through thespecification, are those commonly used in the art. Gene aliases anddescriptions can be found using a variety of online databases, includingGeneCards® (www.genecards.org), HUGO Gene Nomenclature(www.genenames.org), Entrez Gene(www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene), UniProtKB/Swiss-Prot(www.uniprot.org), UniProtKB/TrEMBL (www.uniprot.org), OMIM(www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM), GeneLoc(genecards.weizmann.ac.il/geneloc/), and Ensembl (www.ensembl.org).Generally, gene symbols and names below correspond to those approved byHUGO, and protein names are those recommended by UniProtKB/Swiss-Prot.Common alternatives are provided as well. Where a protein name indicatesa precursor, the mature protein is also implied. Throughout theapplication, gene and protein symbols may be used interchangeably andthe meaning can be derived from context as necessary.

TABLE 4 Illustrative Biomarkers Illustrative Class Biomarkers Drugassociated ABCC1, ABCG2, ACE2, ADA, ADH1C, ADH4, AGT, AR, AREG, ASNS,BCL2, targets and BCRP, BDCA1, beta III tubulin, BIRC5, B-RAF, BRCA1,BRCA2, CA2, caveolin, prognostic CD20, CD25, CD33, CD52, CDA, CDKN2A,CDKN1A, CDKN1B, CDK2, markers CDW52, CES2, CK 14, CK 17, CK 5/6, c-KIT,c-Met, c-Myc, COX-2, Cyclin D1, DCK, DHFR, DNMT1, DNMT3A, DNMT3B,E-Cadherin, ECGF1, EGFR, EML4- ALK fusion, EPHA2, Epiregulin, ER, ERBR2,ERCC1, ERCC3, EREG, ESR1, FLT1, folate receptor, FOLR1, FOLR2, FSHB,FSHPRH1, FSHR, FYN, GART, GNA11, GNAQ, GNRH1, GNRHR1, GSTP1, HCK, HDAC1,hENT-1, Her2/Neu, HGF, HIF1A, HIG1, HSP90, HSP90AA1, HSPCA, IGF-1R,IGFRBP, IGFRBP3, IGFRBP4, IGFRBP5, IL13RA1, IL2RA, KDR, Ki67, KIT,K-RAS, LCK, LTB, Lymphotoxin Beta Receptor, LYN, MET, MGMT, MLH1, MMR,MRP1, MS4A1, MSH2, MSH5, Myc, NFKB1, NFKB2, NFKBIA, NRAS, ODC1, OGFR,p16, p21, p27, p53, p95, PARP-1, PDGFC, PDGFR, PDGFRA, PDGFRB, PGP, PGR,PI3K, POLA, POLA1, PPARG, PPARGC1, PR, PTEN, PTGS2, PTPN12, RAF1, RARA,ROS1, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2,SSTR3, SSTR4, SSTR5, Survivin, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TS,TUBB3, TXN, TXNRD1, TYMS, VDR, VEGF, VEGFA, VEGFC, VHL, YES1, ZAP70 Drugassociated ABL1, STK11, FGFR2, ERBB4, SMARCB1, CDKN2A, CTNNB1, FGFR1,FLT3, targets and NOTCH1, NPM1, SRC, SMAD4, FBXW7, PTEN, TP53, AKT1,ALK, APC, prognostic CDH1, C-Met, HRAS, IDH1, JAK2, MPL, PDGFRA, SMO,VHL, ATM, CSF1R, markers FGFR3, GNAS, ERBB2, HNF1A, JAK3, KDR, MLH1,PTPN11, RB1, RET, c-Kit, EGFR, PIK3CA, NRAS, GNA11, GNAQ, KRAS, BRAFDrug associated ALK, AR, BRAF, cKIT, cMET, EGFR, ER, ERCC1, GNA11, HER2,IDH1, KRAS, targets and MGMT, MGMT promoter methylation, NRAS, PDGFRA,Pgp, PIK3CA, PR, prognostic PTEN, ROS1, RRM1, SPARC, TLE3, TOP2A, TOPO1,TS, TUBB3, VHL markers Drug associated ABL1, AKT1, ALK, APC, AR, ATM,BRAF, BRAF, BRCA1, BRCA2, CDH1, targets cKIT, cMET, CSF1R, CTNNB1, EGFR,EGFR (H-score), EGFRvIII, ER, ERBB2 (HER2), ERBB4, ERCC1, FBXW7, FGFR1,FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, IDH2, JAK2,JAK3, KDR (VEGFR2), KRAS, MGMT, MGMT Promoter Methylation,microsatellite instability (MSI), MLH1, MPL, MSH2, MSH6, NOTCH1, NPM1,NRAS, PD-1, PDGFRA, PD-L1, Pgp, PIK3CA, PMS2, PR, PTEN, PTPN11, RB1,RET, ROS1, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1,TP53, TS, TUBB3, VHL Drug associated 1p19q co-deletion, ABL1, AKT1, ALK,APC, AR, ARAF, ATM, BAP1, BRAF, targets BRCA1, BRCA2, CDH1, CHEK1,CHEK2, cKIT, cMET, CSF1R, CTNNB1, DDR2, EGFR, EGFRvIII, ER, ERBB2(HER2), ERBB3, ERBB4, ERCC1, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ,GNAS, H3K36me3, HER2, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR (VEGFR2),KRAS, MDMT, MGMT, MGMT Methylation, Microsatellite instability, MLH1,MPL, MSH2, MSH6, NF1, NOTCH1, NPM1, NRAS, NY-ESO-1, PD-1, PDGFRA, PD-L1,Pgp, PIK3CA, PMS2, PR, PTEN, PTPN11, RAF1, RB1, RET, ROS1, ROS1, RRM1,SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TRKA, TS,TUBB3, VHL, WT1 Drug associated ABL1, AKT1, ALK, APC, AR, ATM, BRAF,BRAF, BRCA1, BRCA2, CDH1, targets cKIT, cMET, CSF1R, CTNNB1, EGFR, EGFR(H-score), EGFRvIII, ER, ERBB2 (HER2), ERBB4, ERCC1, FBXW7, FGFR1,FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, IDH2, JAK2,JAK3, KDR (VEGFR2), KRAS, MGMT, MGMT Promoter Methylation,microsatellite instability (MSI), MLH1, MPL, MSH2, MSH6, NOTCH1, NPM1,NRAS, PD-1, PDGFRA, PD-L1, Pgp, PIK3CA, PMS2, PR, PTEN, PTPN11, RB1,RET, ROS1, RRM1, SMAD4, SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1,TP53, TS, TUBB3, VHL Drug associated 1p19q, ALK, ALK (2p23), AndrogenReceptor, BRCA, cMET, EGFR, EGFR, targets EGFRvIII, ER, ERCC1, Her2,Her2/Neu, MGMT, MGMT Promoter Methylation, microsatellite instability(MSI), MLH1, MSH2, MSH6, PD-1, PD-L1, PMS2, PR, PTEN, ROS1, RRM1, TLE3,TOP2A, TOP2A, TOPO1, TS, TUBB3 Drug associated TOP2A, Chromosome 17alteration, PBRM1 (PB1/BAF180), BAP1, SETD2 (ANTI- targets HISTONE H3),MDM2, Chromosome 12 alteration, ALK, CTLA4, CD3, NY-ESO- 1, MAGE-A, TP,EGFR 5-aminosalicyclic μ-protocadherin, KLF4, CEBPα acid (5-ASA)efficacy Cancer treatment AR, AREG (Amphiregulin), BRAF, BRCA1, cKIT,cMET, EGFR, EGFR associated w/T790M, EML4-ALK, ER, ERBB3, ERBB4, ERCC1,EREG, GNA11, GNAQ, markers hENT-1, Her2, Her2 Exon 20 insert, IGF1R,Ki67, KRAS, MGMT, MGMT methylation, MSH2, MSI, NRAS, PGP (MDR1), PIK3CA,PR, PTEN, ROS1, ROS1 translocation, RRM1, SPARC, TLE3, TOPO1, TOPO2A,TS, TUBB3, VEGFR2 Cancer treatment AR, AREG, BRAF, BRCA1, cKIT, cMET,EGFR, EGFR w/T790M, EML4-ALK, associated ER, ERBB3, ERBB4, ERCC1, EREG,GNA11, GNAQ, Her2, Her2 Exon 20 insert, markers IGFR1, Ki67, KRAS,MGMT-Me, MSH2, MSI, NRAS, PGP (MDR-1), PIK3CA, PR, PTEN, ROS1translocation, RRM1, SPARC, TLE3, TOPO1, TOPO2A, TS, TUBB3, VEGFR2 Coloncancer AREG, BRAF, EGFR, EML4-ALK, ERCC1, EREG, KRAS, MSI, NRAS, PIK3CA,treatment PTEN, TS, VEGFR2 associated markers Colon cancer AREG, BRAF,EGFR, EML4-ALK, ERCC1, EREG, KRAS, MSI, NRAS, PIK3CA, treatment PTEN,TS, VEGFR2 associated markers Melanoma BRAF, cKIT, ERBB3, ERBB4, ERCC1,GNA11, GNAQ, MGMT, MGMT treatment methylation, NRAS, PIK3CA, TUBB3,VEGFR2 associated markers Melanoma BRAF, cKIT, ERBB3, ERBB4, ERCC1,GNA11, GNAQ, MGMT-Me, NRAS, treatment PIK3CA, TUBB3, VEGFR2 associatedmarkers Ovarian cancer BRCA1, cMET, EML4-ALK, ER, ERBB3, ERCC1, hENT-1,HER2, IGF1R, treatment PGP(MDR1), PIK3CA, PR, PTEN, RRM1, TLE3, TOPO1,TOPO2A, TS associated markers Ovarian cancer BRCA1, cMET, EML4-ALK(translocation), ER, ERBB3, ERCC1, HER2, PIK3CA, treatment PR, PTEN,RRM1, TLE3, TS associated markers Breast cancer BRAF, BRCA1, EGFR, EGFRT790M, EML4-ALK, ER, ERBB3, ERCC1, HER2, treatment Ki67, PGP (MDR1),PIK3CA, PR, PTEN, ROS1, ROS1 translocation, RRM1, associated TLE3,TOPO1, TOPO2A, TS markers Breast cancer BRAF, BRCA1, EGFR w/T790M,EML4-ALK, ER, ERBB3, ERCC1, HER2, Ki67, treatment KRAS, PIK3CA, PR,PTEN, ROS1 translocation, RRM1, TLE3, TOPO1, TOPO2A, associated TSmarkers NSCLC cancer BRAF, BRCA1, cMET, EGFR, EGFR w/T790M, EML4-ALK,ERCC1, Her2 Exon treatment 20 insert, KRAS, MSH2, PIK3CA, PTEN, ROS1(trans), RRM1, TLE3, TS, associated VEGFR2 markers NSCLC cancer BRAF,cMET, EGFR, EGFR w/T790M, EML4-ALK, ERCC1, Her2 Exon 20 insert,treatment KRAS, MSH2, PIK3CA, PTEN, ROS1 translocation, RRM1, TLE3, TSassociated markers Mutated in AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A,c-Kit, C-Met, CSF1R, cancers CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1,FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3,KDR, KRAS, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11,RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL Mutated in ALK,BRAF, BRCA1, BRCA2, EGFR, ERRB2, GNA11, GNAQ, IDH1, IDH2, cancers KIT,KRAS, MET, NRAS, PDGFRA, PIK3CA, PTEN, RET, SRC, TP53 Mutated in AKT1,HRAS, GNAS, MEK1, MEK2, ERK1, ERK2, ERBB3, CDKN2A, PDGFRB, cancersIFG1R, FGFR1, FGFR2, FGFR3, ERBB4, SMO, DDR2, GRB1, PTCH, SHH, PD1,UGT1A1, BIM, ESR1, MLL, AR, CDK4, SMAD4 Mutated in ABL, APC, ATM, CDH1,CSFR1, CTNNB1, FBXW7, FLT3, HNF1A, JAK2, cancers JAK3, KDR, MLH1, MPL,NOTCH1, NPM1, PTPN11, RB1, SMARCB1, STK11, VHL Mutated in ABL1, AKT1,AKT2, AKT3, ALK, APC, AR, ARAF, ARFRP1, ARID1A, ARID2, cancers ASXL1,ATM, ATR, ATRX, AURKA, AURKB, AXL, BAP1, BARD1, BCL2, BCL2L2, BCL6,BCOR, BCORL1, BLM, BRAF, BRCA1, BRCA2, BRIP1, BTK, CARD11, CBFB, CBL,CCND1, CCND2, CCND3, CCNE1, CD79A, CD79B, CDC73, CDH1, CDK12, CDK4,CDK6, CDK8, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CEBPA, CHEK1, CHEK2, CIC,CREBBP, CRKL, CRLF2, CSF1R, CTCF, CTNNA1, CTNNB1, DAXX, DDR2, DNMT3A,DOT1L, EGFR, EMSY (C11orf30), EP300, EPHA3, EPHA5, EPHB1, ERBB2, ERBB3,ERBB4, ERG, ESR1, EZH2, FAM123B (WTX), FAM46C, FANCA, FANCC, FANCD2,FANCE, FANCF, FANCG, FANCL, FBXW7, FGF10, FGF14, FGF19, FGF23, FGF3,FGF4, FGF6, FGFR1, FGFR2, FGFR3, FGFR4, FLT1, FLT3, FLT4, FOXL2, GATA1,GATA2, GATA3, GID4 (C17orf39), GNA11, GNA13, GNAQ, GNAS, GPR124, GRIN2A,GSK3B, HGF, HRAS, IDH1, IDH2, IGF1R, IKBKE, IKZF1, IL7R, INHBA, IRF4,IRS2, JAK1, JAK2, JAK3, JUN, KAT6A (MYST3), KDM5A, KDM5C, KDM6A, KDR,KEAP1, KIT, KLHL6, KRAS, LRP1B, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MCL1,MDM2, MDM4, MED12, MEF2B, MEN1, MET, MITF, MLH1, MLL, MLL2, MPL, MRE11A,MSH2, MSH6, MTOR, MUTYH, MYC, MYCL1, MYCN, MYD88, NF1, NF2, NFE2L2,NFKBIA, NKX2- 1, NOTCH1, NOTCH2, NPM1, NRAS, NTRK1, NTRK2, NTRK3, NUP93,PAK3, PALB2, PAX5, PBRM1, PDGFRA, PDGFRB, PDK1, PIK3CA, PIK3CG, PIK3R1,PIK3R2, PPP2R1A, PRDM1, PRKAR1A, PRKDC, PTCH1, PTEN, PTPN11, RAD50,RAD51, RAF1, RARA, RB1, RET, RICTOR, RNF43, RPTOR, RUNX1, SETD2, SF3B1,SMAD2, SMAD4, SMARCA4, SMARCB1, SMO, SOCS1, SOX10, SOX2, SPEN, SPOP,SRC, STAG2, STAT4, STK11, SUFU, TET2, TGFBR2, TNFAIP3, TNFRSF14, TOP1,TP53, TSC1, TSC2, TSHR, VHL, WISP3, WT1, XPO1, ZNF217, ZNF703 Gene ALK,BCR, BCL2, BRAF, EGFR, ETV1, ETV4, ETV5, ETV6, EWSR1, MLL, rearrangementin MYC, NTRK1, PDGFRA, RAF1, RARA, RET, ROS1, TMPRSS2 cancer CancerRelated ABL1, ACE2, ADA, ADH1C, ADH4, AGT, AKT1, AKT2, AKT3, ALK, APC,AR, ARAF, AREG, ARFRP1, ARID1A, ARID2, ASNS, ASXL1, ATM, ATR, ATRX,AURKA, AURKB, AXL, BAP1, BARD1, BCL2, BCL2L2, BCL6, BCOR, BCORL1, BCR,BIRC5 (survivin), BLM, BRAF, BRCA1, BRCA2, BRIP1, BTK, CA2, CARD11, CAV,CBFB, CBL, CCND1, CCND2, CCND3, CCNE1, CD33, CD52 (CDW52), CD79A, CD79B,CDC73, CDH1, CDK12, CDK2, CDK4, CDK6, CDK8, CDKN1B, CDKN2A, CDKN2B,CDKN2C, CEBPA, CES2, CHEK1, CHEK2, CIC, CREBBP, CRKL, CRLF2, CSF1R,CTCF, CTNNA1, CTNNB1, DAXX, DCK, DDR2, DHFR, DNMT1, DNMT3A, DNMT3B,DOT1L, EGFR, EMSY (C11orf30), EP300, EPHA2, EPHA3, EPHA5, EPHB1, ERBB2,ERBB3, ERBB4, ERBR2 (typo?), ERCC3, EREG, ERG, ESR1, ETV1, ETV4, ETV5,ETV6, EWSR1, EZH2, FAM123B (WTX), FAM46C, FANCA, FANCC, FANCD2, FANCE,FANCF, FANCG, FANCL, FBXW7, FGF10, FGF14, FGF19, FGF23, FGF3, FGF4,FGF6, FGFR1, FGFR2, FGFR3, FGFR4, FLT1, FLT3, FLT4, FOLR1, FOLR2, FOXL2,FSHB, FSHPRH1, FSHR, GART, GATA1, GATA2, GATA3, GID4 (C17orf39), GNA11,GNA13, GNAQ, GNAS, GNRH1, GNRHR1, GPR124, GRIN2A, GSK3B, GSTP1, HDAC1,HGF, HIG1, HNF1A, HRAS, HSPCA (HSP90), IDH1, IDH2, IGF1R, IKBKE, IKZF1,IL13RA1, IL2, IL2RA (CD25), IL7R, INHBA, IRF4, IRS2, JAK1, JAK2, JAK3,JUN, KAT6A (MYST3), KDM5A, KDM5C, KDM6A, KDR (VEGFR2), KEAP1, KIT,KLHL6, KRAS, LCK, LRP1B, LTB, LTBR, MAP2K1, MAP2K2, MAP2K4, MAP3K1,MAPK, MCL1, MDM2, MDM4, MED12, MEF2B, MEN1, MET, MGMT, MITF, MLH1, MLL,MLL2, MPL, MRE11A, MS4A1 (CD20), MSH2, MSH6, MTAP, MTOR, MUTYH, MYC,MYCL1, MYCN, MYD88, NF1, NF2, NFE2L2, NFKB1, NFKB2, NFKBIA, NGF, NKX2-1,NOTCH1, NOTCH2, NPM1, NRAS, NTRK1, NTRK2, NTRK3, NUP93, ODC1, OGFR,PAK3, PALB2, PAX5, PBRM1, PDGFC, PDGFRA, PDGFRB, PDK1, PGP, PGR (PR),PIK3CA, PIK3CG, PIK3R1, PIK3R2, POLA, PPARG, PPARGC1, PPP2R1A, PRDM1,PRKAR1A, PRKDC, PTCH1, PTEN, PTPN11, RAD50, RAD51, RAF1, RARA, RB1, RET,RICTOR, RNF43, ROS1, RPTOR, RRM1, RRM2, RRM2B, RUNX1, RXR, RXRB, RXRG,SETD2, SF3B1, SMAD2, SMAD4, SMARCA4, SMARCB1, SMO, SOCS1, SOX10, SOX2,SPARC, SPEN, SPOP, SRC, SST, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, STAG2,STAT4, STK11, SUFU, TET2, TGFBR2, TK1, TLE3, TMPRSS2, TNF, TNFAIP3,TNFRSF14, TOP1, TOP2, TOP2A, TOP2B, TP53, TS, TSC1, TSC2, TSHR, TUBB3,TXN, TYMP, VDR, VEGF (VEGFA), VEGFC, VHL, WISP3, WT1, XDH, XPO1, YES1,ZAP70, ZNF217, ZNF703 Cancer Related 5T4, ABI1, ABL1, ABL2, ACKR3,ACSL3, ACSL6, ACVR1B, ACVR2A, AFF1, AFF3, AFF4, AKAP9, AKT1, AKT2, AKT3,ALDH2, ALK, AMER1, ANG1/ANGPT1/TM7SF2, ANG2/ANGPT2/VPS51, APC, AR, ARAF,ARFRP1, ARHGAP26, ARHGEF12, ARID1A, ARID1B, ARID2, ARNT, ASPSCR1, ASXL1,ATF1, ATIC, ATM, ATP1A1, ATP2B3, ATR, ATRX, AURKA, AURKB, AXIN1, AXL,BAP1, BARD1, BBC3, BCL10, BCL11A, BCL11B, BCL2, BCL2L1, BCL2L11, BCL2L2,BCL3, BCL6, BCL7A, BCL9, BCOR, BCORL1, BCR, BIRC3, BLM, BMPR1A, BRAF,BRCA1, BRCA2, BRD3, BRD4, BRIP1, BTG1, BTK, BUB1B, c-KIT, C11orf30,c15orf21, C15orf65, C2orf44, CA6, CACNA1D, CALR, CAMTA1, CANT1, CARD11,CARS, CASC5, CASP8, CBFA2T3, CBFB, CBL, CBLB, CBLC, CCDC6, CCNB1IP1,CCND1, CCND2, CCND3, CCNE1, CD110, CD123, CD137, CD19, CD20, CD274,CD27L, CD38, CD4, CD74, CD79A, CD79B, CDC73, CDH1, CDH11, CDK12, CDK4,CDK6, CDK7, CDK8, CDK9, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CDX2,CEBPA, CHCHD7, CHD2, CHD4, CHEK1, CHEK2, CHIC2, Chk1, CHN1, CIC, CIITA,CLP1, CLTC, CLTCL1, CNBP, CNOT3, CNTRL, COL1A1, COPB1, CoREST, COX6C,CRAF, CREB1, CREB3L1, CREB3L2, CREBBP, CRKL, CRLF2, CRTC1, CRTC3, CSF1R,CSF3R, CTCF, CTLA4, CTNNA1, CTNNB1, CUL3, CXCR4, CYLD, CYP17A1, CYP2D6,DAXX, DDB2, DDIT3, DDR1, DDR2, DDX10, DDX5, DDX6, DEK, DICER1, DLL-4,DNAPK, DNM2, DNMT3A, DOT1L, EBF1, ECT2L, EGFR, EIF4A2, ELF4, ELK4, ELL,ELN, EML4, EP300, EPHA3, EPHA5, EPHA7, EPHA8, EPHB1, EPHB2, EPS15,ERBB2, ERBB3, ERBB4, ERC1, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERG,ERRFI1, ESR1, ETBR, ETV1, ETV4, ETV5, ETV6, EWSR1, EXT1, EXT2, EZH2,EZR, FAK, FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, FAS,FAT1, FBXO11, FBXW7, FCRL4, FEV, FGF10, FGF14, FGF19, FGF2, FGF23, FGF3,FGF4, FGF6, FGFR1, FGFR1OP, FGFR2, FGFR3, FGFR4, FH, FHIT, FIP1L1,FKBP12, FLCN, FLI1, FLT1, FLT3, FLT4, FNBP1, FOXA1, FOXL2, FOXO1, FOXO3,FOXO4, FOXP1, FRS2, FSTL3, FUBP1, FUS, GABRA6, GAS7, GATA1, GATA2,GATA3, GATA4, GATA6, GID4, GITR, GLI1, GMPS, GNA11, GNA13, GNAQ, GNAS,GNRH1, GOLGA5, GOPC, GPC3, GPHN, GPR124, GRIN2A, GRM3, GSK3B, GUCY2C,H3F3A, H3F3B, HCK, HERPUD1, HEY1, HGF, HIP1, HIST1H3B, HIST1H4I, HLF,HMGA1, HMGA2, HMT, HNF1A, HNRNPA2B1, HOOK3, HOXA11, HOXA13, HOXA9,HOXC11, HOXC13, HOXD11, HOXD13, HRAS, HSD3B1, HSP90AA1, HSP90AB1, IAP,IDH1, IDH2, IGF1R, IGF2, IKBKE, IKZF1, IL2, IL21R, IL6, IL6ST, IL7R,INHBA, INPP4B, IRF2, IRF4, IRS2, ITGAV, ITGB1, ITK, JAK1, JAK2, JAK3,JAZF1, JUN, KAT6A, KAT6B, KCNJ5, KDM5A, KDM5C, KDM6A, KDR, KDSR, KEAP1,KEL, KIAA1549, KIF5B, KIR3DL1, KLF4, KLHL6, KLK2, KMT2A, KMT2C, KMT2D,KRAS, KTN1, LASP1, LCK, LCP1, LGALS3, LGR5, LHFP, LIFR, LMO1, LMO2,LOXL2, LPP, LRIG3, LRP1B, LSD1, LYL1, LYN, LZTR1, MAF, MAFB, MAGI2,MALT1, MAML2, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAPK1, MAPK11, MAX, MCL1,MDM2, MDM4, MDS2, MECOM, MED12, MEF2B, MEK1, MEK2, MEN1, MET, MITF,MKL1, MLF1, MLH1, MLLT1, MLLT10, MLLT11, MLLT3, MLLT4, MLLT6, MMP9, MN1,MNX1, MPL, MPS1, MRE11A, MS4A1, MSH2, MSH6, MSI2, MSN, MST1R, MTCP1,MTOR, MUC1, MUC16, MUTYH, MYB, MYC, MYCL, MYCN, MYD88, MYH11, MYH9,NACA, NAE1, NBN, NCKIPSD, NCOA1, NCOA2, NCOA4, NDRG1, NF1, NF2, NFE2L2,NFIB, NFKB2, NFKBIA, NIN, NKX2-1, NONO, NOTCH1, NOTCH2, NOTCH3, NPM1,NR4A3, NRAS, NSD1, NT5C2, NTRK1, NTRK2, NTRK3, NUMA1, NUP214, NUP93,NUP98, NUTM1, NUTM2B, OLIG2, OMD, P2RY8, PAFAH1B2, PAK3, PALB2, PARK2,PARP1, PATZ1, PAX3, PAX5, PAX7, PAX8, PBRM1, PBX1, PCM1, PCSK7, PDCD1,PDCD1LG2, PDE4DIP, PDGFB, PDGFRA, PDGFRB, PDK1, PER1, PHF6, PHOX2B,PICALM, PIK3C2B, PIK3CA, PIK3CB, PIK3CD, PIK3CG, PIK3R1, PIK3R2, PIM1,PKC, PLAG1, PLCG2, PML, PMS1, PMS2, POLD1, POLE, POT1, POU2AF1, POU5F1,PPARG, PPP2R1A, PRCC, PRDM1, PRDM16, PREX2, PRF1, PRKAR1A, PRKCI, PRKDC,PRLR, PRRX1, PRSS8, PSIP1, PTCH1, PTEN, PTK2, PTPN11, PTPRC, PTPRD, QKI,RABEP1, RAC1, RAD21, RAD50, RAD51, RAD51B, RAF1, RALGDS, RANBP17,RANBP2, RANKL, RAP1GDS1, RARA, RB1, RBM10, RBM15, RECQL4, REL, RET,RHOH, RICTOR, RMI2, RNF213, RNF43, ROS1, RPL10, RPL20, RPL5, RPN1,RPS6KB1, RPTOR, RUNX1, RUNx1T1, SBDS, SDC4, SDHA, SDHAF2, SDHB, SDHC,SDHD, SEPT5, SEPT6, SEPT9, SET, SETBP1, SETD2, SF3B1, SFPQ, SH2B3,SH3GL1, SLAMF7, SLC34A2, SLC45A3, SLIT2, SMAD2, SMAD3, SMAD4, SMARCA4,SMARCB1, SMARCE1, SMO, SNCAIP, SNX29, SOCS1, SOX10, SOX2, SOX9, SPECC1,SPEN, SPOP, SPTA1, SRC, SRGAP3, SRSF2, SRSF3, SS18, SS18L1, SSX1, SSX2,SSX4, STAG2, STAT3, STAT4, STAT5B, STEAP1, STIL, STK11, SUFU, SUZ12,SYK, TAF1, TAF15, TAL1, TAL2, TBL1XR1, TBX3, TCEA1, TCF12, TCF3, TCF7L2,TCL1A, TERC, TERT, TET1, TET2, TFE3, TFEB, TFG, TFPT, TFRC, TGFB1,TGFBR2, THRAP3, TIE2, TLX1, TLX3, TMPRSS2, TNFAIP3, TNFRSF14, TNFRSF17,TOP1, TOP2A, TP53, TPM3, TPM4, TPR, TRAF7, TRIM26, TRIM27, TRIM33,TRIP11, TRRAP, TSC1, TSC2, TSHR, TTL, U2AF1, UBA1, UBR5, USP6, VEGFA,VEGFB, VEGFR, VHL, VTI1A, WAS, WEE1, WHSC1, WHSC1L1, WIF1, WISP3, WNT11,WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT6, WNT7B, WRN, WT1, WWTR1, XPA, XPC,XPO1, YWHAE, ZAK, ZBTB16, ZBTB2, ZMYM2, ZNF217, ZNF331, ZNF384, ZNF521,ZNF703, ZRSR2 Cancer Related ABL2, ACSL3, ACSL6, AFF1, AFF3, AFF4,AKAP9, AKT3, ALDH2, APC, ARFRP1, ARHGAP26, ARHGEF12, ARID2, ARNT,ASPSCR1, ASXL1, ATF1, ATIC, ATM, ATP1A1, ATR, AURKA, AXIN1, AXL, BAP1,BARD1, BCL10, BCL11A, BCL2L11, BCL3, BCL6, BCL7A, BCL9, BCR, BIRC3, BLM,BMPR1A, BRAF, BRCA1, BRCA2, BRIP1, BUB1B, C11orf30, C2orf44, CACNA1D,CALR, CAMTA1, CANT1, CARD11, CARS, CASC5, CASP8, CBFA2T3, CBFB, CBL,CBLB, CCDC6, CCNB1IP1, CCND2, CD274, CD74, CD79A, CDC73, CDH11, CDKN1B,CDX2, CHEK1, CHEK2, CHIC2, CHN1, CIC, CIITA, CLP1, CLTC, CLTCL1, CNBP,CNTRL, COPB1, CREB1, CREB3L1, CREB3L2, CRTC1, CRTC3, CSF1R, CSF3R, CTCF,CTLA4, CTNNA1, CTNNB1, CYLD, CYP2D6, DAXX, DDR2, DDX10, DDX5, DDX6, DEK,DICER1, DOT1L, EBF1, ECT2L, ELK4, ELL, EML4, EPHA3, EPHA5, EPHB1, EPS15,ERBB3, ERBB4, ERC1, ERCC2, ERCC3, ERCC4, ERCC5, ERG, ESR1, ETV1, ETV5,ETV6, EWSR1, EXT1, EXT2, EZR, FANCA, FANCC, FANCD2, FANCE, FANCG, FANCL,FAS, FBXO11, FBXW7, FCRL4, FGF14, FGF19, FGF23, FGF6, FGFR1OP, FGFR4,FH, FHIT, FIP1L1, FLCN, FLI1, FLT1, FLT3, FLT4, FNBP1, FOXA1, FOXO1,FOXP1, FUBP1, FUS, GAS7, GID4, GMPS, GNA13, GNAQ, GNAS, GOLGA5, GOPC,GPHN, GPR124, GRIN2A, GSK3B, H3F3A, H3F3B, HERPUD1, HGF, HIP1, HMGA1,HMGA2, HNRNPA2B1, HOOK3, HSP90AA1, HSP90AB1, IDH1, IDH2, IGF1R, IKZF1,IL2, IL21R, IL6ST, IL7R, IRF4, ITK, JAK1, JAK2, JAK3, JAZF1, KDM5A,KEAP1, KIAA1549, KIF5B, KIT, KLHL6, KMT2A, KMT2C, KMT2D, KRAS, KTN1,LCK, LCP1, LGR5, LHFP, LIFR, LPP, LRIG3, LRP1B, LYL1, MAF, MALT1, MAML2,MAP2K2, MAP2K4, MAP3K1, MDM4, MDS2, MEF2B, MEN1, MITF, MLF1, MLH1,MLLT1, MLLT10, MLLT3, MLLT4, MLLT6, MNX1, MRE11A, MSH2, MSH6, MSI2,MTOR, MYB, MYCN, MYD88, MYH11, MYH9, NACA, NCKIPSD, NCOA1, NCOA2, NCOA4,NF1, NFE2L2, NFIB, NFKB2, NIN, NOTCH2, NPM1, NR4A3, NSD1, NT5C2, NTRK2,NTRK3, NUP214, NUP93, NUP98, NUTM1, PALB2, PAX3, PAX5, PAX7, PBRM1,PBX1, PCM1, PCSK7, PDCD1, PDCD1LG2, PDGFB, PDGFRA, PDGFRB, PDK1, PER1,PICALM, PIK3CA, PIK3R1, PIK3R2, PIM1, PML, PMS2, POLE, POT1, POU2AF1,PPARG, PRCC, PRDM1, PRDM16, PRKAR1A, PRRX1, PSIP1, PTCH1, PTEN, PTPN11,PTPRC, RABEP1, RAC1, RAD50, RAD51, RAD51B, RAF1, RALGDS, RANBP17,RAP1GDS1, RARA, RBM15, REL, RET, RMI2, RNF43, RPL20, RPL5, RPN1, RPTOR,RUNX1, RUNX1T1, SBDS, SDC4, SDHAF2, SDHB, SDHC, SDHD, 8-Sep, SET,SETBP1, SETD2, SF3B1, SH2B3, SH3GL1, SLC34A2, SMAD2, SMAD4, SMARCB1,SMARCE1, SMO, SNX29, SOX10, SPECC1, SPEN, SRGAP3, SRSF2, SRSF3, SS18,SS18L1, STAT3, STAT4, STAT5B, STIL, STK11, SUFU, SUZ12, SYK, TAF15,TCF12, TCF3, TCF7L2, TET1, TET2, TFEB, TFG, TFRC, TGFBR2, TLX1, TNFAIP3,TNFRSF14, TNFRSF17, TP53, TPM3, TPM4, TPR, TRAF7, TRIM26, TRIM27,TRIM33, TRIP11, TRRAP, TSC1, TSC2, TSHR, TTL, U2AF1, USP6, VEGFA, VEGFB,VTI1A, WHSC1, WHSC1L1, WIF1, WISP3, WRN, WWTR1, XPA, XPC, XPO1, YWHAE,ZMYM2, ZNF217, ZNF331, ZNF384, ZNF521, ZNF703 Gene fusions and AKT3,ALK, ARHGAP26, AXL, BRAF, BRD3/4, EGFR, ERG, ESR1, ETV1/4/5/6, mutationsin EWSR1, FGFR1, FGFR2, FGFR3, FGR, INSR, MAML2, MAST1/2, MET, MSMB,cancer MUSK, MYB, NOTCH1/2, NRG1, NTRK1/2/3, NUMBL, NUTM1, PDGFRA/B,PIK3CA, PKN1, PPARG, PRKCA/B, RAF1, RELA, RET, ROS1, RSPO2/3, TERT,TFE3, TFEB, THADA, TMPRSS2 Gene fusions and ABL1 fusion to (ETV6,NUP214, RCSD1, RANBP2, SNX2, or ZMIZ1); ABL2 mutations in fusion to(PAG1 or RCSD1); CSF1R fusion to (SSBP2); PDGFRB fusion to (EBF1, cancerSSBP2, TNIP1 or ZEB2); CRLF2 fusion to (P2RY8); JAK2 fusion to (ATF7IP,BCR, ETV6, PAX5, PPFIBP1, SSBP2, STRN3, TERF2, or TPR); EPOR fusion to(IGH or IGK); IL2RB fusion to (MYH9); NTRK3 fusion to (ETV6); PTK2Bfusion to (KDM6A or STAG2); TSLP fusion to (IQGAP2); TYK2 fusion to(MYB) Cytohesions cytohesin-1 (CYTH1), cytohesin-2 (CYTH2; ARNO),cytohesin-3 (CYTH3; Grp1; ARNO3), cytohesin-4 (CYTH4) Cancer/Angio Erb2, Erb 3, Erb 4, UNC93a, B7H3, MUC1, MUC2, MUC16, MUC17, 5T4, RAGE, VEGFA, VEGFR2, FLT1, DLL4, Epcam Tissue (Breast) BIG H3, GCDFP-15, PR(B),GPR 30, CYFRA 21, BRCA 1, BRCA 2, ESR 1, ESR2 Tissue (Prostate) PSMA,PCSA, PSCA, PSA, TMPRSS2 Inflammation/ MFG-E8, IFNAR, CD40, CD80, MICB,HLA-DRb, IL-17-Ra Immune

Examples of additional biomarkers that can be incorporated into themethods and compositions of the invention include without limitationthose disclosed in International Patent Application Nos.PCT/US2009/62880, filed Oct. 30, 2009; PCT/US2009/006095, filed Nov. 12,2009; PCT/US2011/26750, filed Mar. 1, 2011; PCT/US2011/031479, filedApr. 6, 2011; PCT/US11/48327, filed Aug. 18, 2011; PCT/US2008/71235,filed Jul. 25, 2008; PCT/US10/58461, filed Nov. 30, 2010;PCT/US2011/21160, filed Jan. 13, 2011; PCT/US2013/030302, filed Mar. 11,2013; PCT/US12/25741, filed Feb. 17, 2012; PCT/2008/76109, filed Sep.12, 2008; PCT/US12/42519, filed Jun. 14, 2012; PCT/US12/50030, filedAug. 8, 2012; PCT/US12/49615, filed Aug. 3, 2012; PCT/US12/41387, filedJun. 7, 2012; PCT/US2013/072019, filed Nov. 26, 2013; PCT/US2014/039858,filed May 28, 2013; PCT/IB2013/003092, filed Oct. 23, 2013;PCT/US13/76611, filed Dec. 19, 2013; PCT/US14/53306, filed Aug. 28,2014; and PCT/US15/62184, filed Nov. 23, 2015; PCT/US16/40157, filedJun. 29, 2016; PCT/US16/44595, filed Jul. 28, 2016; and PCT/US16/21632,filed Mar. 9, 2016; each of which applications is incorporated herein byreference in its entirety.

In various embodiments of the invention, the biomarkers or biosignatureused to detect or assess any of the conditions or diseases disclosedherein can comprise one or more biomarkers in one of several differentcategories of markers, wherein the categories include without limitationone or more of: 1) disease specific biomarkers; 2) cell- ortissue-specific biomarkers; 3) vesicle-specific markers (e.g., generalvesicle biomarkers); 4) angiogenesis-specific biomarkers; and 5)immunomodulatory biomarkers. Examples of all such markers are disclosedherein and known to a person having ordinary skill in the art.Furthermore, a biomarker known in the art that is characterized to havea role in a particular disease or condition can be adapted for use as atarget in compositions and methods of the invention. In furtherembodiments, such biomarkers of interest may be cellular or vesicularsurface markers, or a combination of surface markers and soluble orpayload markers (e.g., molecules enclosed by a microvesicle). Thebiomarkers assessed can be from a combination of sources. For example, adisease or disorder may be detected or characterized by assessing acombination of proteins, nucleic acids, vesicles, circulatingbiomarkers, biomarkers from a tissue sample, and the like. In addition,as noted herein, the biological sample assessed can be any biologicalfluid, or can comprise individual components present within suchbiological fluid (e.g., vesicles, nucleic acids, proteins, or complexesthereof).

Biomarker Detection

The compositions and methods of the invention can be used to assess anyuseful biomarkers in a biological sample for charactering a phenotypeassociated with the sample. Such biomarkers include all sorts ofbiological entities such as proteins, nucleic acids, lipids,carbohydrates, complexes of any thereof, and microvesicles.

The aptamers of the invention can be used to provide a biosignature intissue or bodily fluids, e.g., by assessing various biomarkers therein.See, e.g., FIGS. 10B-C. The aptamers of the invention can also be usedto assess levels or presence of their specific target molecule. See,e.g., FIG. 10A. In addition, aptamers of the invention are used tocapture or isolated a component present in a biological sample that hasthe aptamer's target molecule present. For example, if a given surfaceantigen is present on a cell, cell fragment or cell-derivedextracellular vesicle, a binding agent to the biomarker, includingwithout limitation an aptamer provided by the invention, may be used tocapture or isolate the cell, cell fragment or cell-derived extracellularvesicles. See, e.g., FIGS. 1A-B, 10A. Such captured or isolated entitiesmay be further characterized to assess additional surface antigens orinternal “payload” molecules, e.g., nucleic acid molecules, lipids,sugars, polypeptides or functional fragments thereof, or anything elsepresent in the cellular milieu that may be used as a biomarker.Therefore, aptamers of the invention are used not only to assess one ormore surface antigen of interest but are also used to separate acomponent present in a biological sample, where the componentsthemselves can be comprised within the biosignature.

The methods of the invention can comprise multiplex analysis of at least2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or moredifferent biomarkers. For example, an oligonucleotide pool may containany number of individual aptamers that can target different biomarkers.As another example, an assay can be performed with a plurality ofparticles that are differentially labeled. There can be at least 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75or 100 differentially labeled particles. The particles may be externallylabeled, such as with a tag, or they may be intrinsically labeled. Eachdifferentially labeled particle can be coupled to a capture agent, suchas a antibody or aptamer, and can be used to capture its target. Themultiple capture agents can be selected to characterize a phenotype ofinterest, including capture agents against general vesicle biomarkers,cell-of-origin specific biomarkers, and disease biomarkers. One or morecaptured biomarkers can be detected by a plurality of binding agents.The binding agent can be directly labeled to facilitate detection.Alternatively, the binding agent is labeled by a secondary agent. Forexample, the binding agent may be an antibody or aptamer for abiomarker, wherein the binding agent is linked to biotin. A secondaryagent comprises streptavidin linked to a reporter and can be added todetect the biomarker. In some embodiments, the captured vesicle isassayed for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 50, 75 or 100 different biomarkers. For example,multiple detectors, i.e., detection of multiple biomarkers of a capturedvesicle or population of vesicles, can increase the signal obtained,permitted increased sensitivity, specificity, or both, and the use ofsmaller amounts of samples. Detection can be with more than onebiomarker, including without limitation more than one vesicle markersuch as in any of Tables 3-4, and Tables 10-17.

An immunoassay based method (e.g., sandwich assay) can be used to detecta biomarker of interest. An example includes ELISA. A binding agent canbe bound to a well. For example, a binding agent such as an aptamer orantibody to biomarker of interest can be attached to a well. A capturedbiomarker can be detected based on the methods described herein. FIG. 1Ashows an illustrative schematic for a sandwich-type of immunoassay. Thecapture agent can be against a cellular or vesicular antigen of. In thefigure, the captured entities are detected using fluorescently labeledbinding agent (detection agent) against antigens of interest. Multiplecapture binding agents can be used, e.g., in distinguishable addresseson an array or different wells of an immunoassay plate. The detectionbinding agents can be against the same antigen as the capture bindingagent, or can be directed against other markers. The capture bindingagent can be any useful binding agent, e.g., tethered aptamers,antibodies or lectins, and/or the detector antibodies can be similarlysubstituted, e.g., with detectable (e.g., labeled) aptamers, antibodies,lectins or other binding proteins or entities.

In an embodiment, one or more capture agents to a general vesiclebiomarker, a cell-of-origin marker, and/or a disease marker are usedalong with detection agents against general vesicle biomarker, such astetraspanin molecules including without limitation one or more of CD9,CD63 and CD81, or other markers in Table 3 herein. Examples ofmicrovesicle surface antigens are disclosed herein, e.g. in Tables 3-4and 10-17. Further biomarkers and detection techniques are disclosed inInternational Patent Application Nos. PCT/US2009/62880, filed Oct. 30,2009; PCT/US2009/006095, filed Nov. 12, 2009; PCT/US2011/26750, filedMar. 1, 2011; PCT/US2011/031479, filed Apr. 6, 2011; PCT/US11/48327,filed Aug. 18, 2011; PCT/US2008/71235, filed Jul. 25, 2008;PCT/US10/58461, filed Nov. 30, 2010; PCT/US2011/21160, filed Jan. 13,2011; PCT/US2013/030302, filed Mar. 11, 2013; PCT/US12/25741, filed Feb.17, 2012; PCT/2008/76109, filed Sep. 12, 2008; PCT/US12/42519, filedJun. 14, 2012; PCT/US12/50030, filed Aug. 8, 2012; PCT/US12/49615, filedAug. 3, 2012; PCT/US12/41387, filed Jun. 7, 2012; PCT/US2013/072019,filed Nov. 26, 2013; PCT/US2014/039858, filed May 28, 2013;PCT/IB2013/003092, filed Oct. 23, 2013; PCT/US13/76611, filed Dec. 19,2013; PCT/US14/53306, filed Aug. 28, 2014; PCT/US15/62184, filed Nov.23, 2015; PCT/US16/40157, filed Jun. 29, 2016; PCT/US16/44595, filedJul. 28, 2016; and PCT/US16/21632, filed Mar. 9, 2016; each of whichapplications is incorporated herein by reference in its entirety.

Techniques of detecting biomarkers or capturing sample components usingan aptamer of the invention include the use of a planar substrate suchas an array (e.g., biochip or microarray), with molecules immobilized tothe substrate as capture agents that facilitate the detection of aparticular biosignature. The array can be provided as part of a kit forassaying one or more biomarkers. Aptamers of the invention can beincluded in an array for detection and diagnosis of diseases includingpresymptomatic diseases. In some embodiments, an array comprises acustom array comprising biomolecules selected to specifically identifybiomarkers of interest. Customized arrays can be modified to detectbiomarkers that increase statistical performance, e.g., additionalbiomolecules that identifies a biosignature which lead to improvedcross-validated error rates in multivariate prediction models (e.g.,logistic regression, discriminant analysis, or regression tree models).In some embodiments, customized array(s) are constructed to study thebiology of a disease, condition or syndrome and profile biosignatures indefined physiological states. Markers for inclusion on the customizedarray be chosen based upon statistical criteria, e.g., having a desiredlevel of statistical significance in differentiating between phenotypesor physiological states. In some embodiments, standard significance ofp-value=0.05 is chosen to exclude or include biomolecules on themicroarray. The p-values can be corrected for multiple comparisons. Asan illustrative example, nucleic acids extracted from samples from asubject with or without a disease can be hybridized to a high densitymicroarray that binds to thousands of gene sequences. Nucleic acidswhose levels are significantly different between the samples with orwithout the disease can be selected as biomarkers to distinguish samplesas having the disease or not. A customized array can be constructed todetect the selected biomarkers. In some embodiments, customized arrayscomprise low density microarrays, which refer to arrays with lowernumber of addressable binding agents, e.g., tens or hundreds instead ofthousands. Low density arrays can be formed on a substrate. In someembodiments, customizable low density arrays use PCR amplification inplate wells, e.g., TaqMan® Gene Expression Assays (Applied Biosystems byLife Technologies Corporation, Carlsbad, Calif.).

An aptamer of the invention or other useful binding agent may be linkeddirectly or indirectly to a solid surface or substrate. A solid surfaceor substrate can be any physically separable solid to which a bindingagent can be directly or indirectly attached including, but not limitedto, surfaces provided by microarrays and wells, particles such as beads,columns, optical fibers, wipes, glass and modified or functionalizedglass, quartz, mica, diazotized membranes (paper or nylon),polyformaldehyde, cellulose, cellulose acetate, paper, ceramics, metals,metalloids, semiconductive materials, quantum dots, coated beads orparticles, other chromatographic materials, magnetic particles; plastics(including acrylics, polystyrene, copolymers of styrene or othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes,Teflon material, etc.), polysaccharides, nylon or nitrocellulose,resins, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, plastics, ceramics,conducting polymers (including polymers such as polypyrrole andpolyindole); micro or nanostructured surfaces such as nucleic acidtiling arrays, nanotube, nanowire, or nanoparticulate decoratedsurfaces; or porous surfaces or gels such as methacrylates, acrylamides,sugar polymers, cellulose, silicates, or other fibrous or strandedpolymers. In addition, as is known the art, the substrate may be coatedusing passive or chemically-derivatized coatings with any number ofmaterials, including polymers, such as dextrans, acrylamides, gelatinsor agarose. Such coatings can facilitate the use of the array with abiological sample.

An aptamer or other useful binding agent can be conjugated to adetectable entity or label. Appropriate labels include withoutlimitation a magnetic label, a fluorescent moiety, an enzyme, achemiluminescent probe, a metal particle, a non-metal colloidalparticle, a polymeric dye particle, a pigment molecule, a pigmentparticle, an electrochemically active species, semiconductor nanocrystalor other nanoparticles including quantum dots or gold particles,fluorophores, quantum dots, or radioactive labels. Protein labelsinclude green fluorescent protein (GFP) and variants thereof (e.g., cyanfluorescent protein and yellow fluorescent protein); and luminescentproteins such as luciferase, as described below. Radioactive labelsinclude without limitation radioisotopes (radionuclides), such as ³H,¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I,¹³¹I, ¹³³Xe, ¹⁷⁷Lu, ²¹¹At, or ²¹³Bi. Fluorescent labels include withoutlimitation a rare earth chelate (e.g., europium chelate), rhodamine;fluorescein types including without limitation FITC,5-carboxyfluorescein, 6-carboxy fluorescein; a rhodamine type includingwithout limitation TAMRA; dansyl; Lissamine; cyanines; phycoerythrins;Texas Red; Cy3, Cy5, dapoxyl, NBD, Cascade Yellow, dansyl, PyMPO,pyrene, 7-diethylaminocoumarin-3-carboxylic acid and other coumarinderivatives, Marina Blue™, Pacific Blue™, Cascade Blue™,2-anthracenesulfonyl, PyMPO, 3,4,9,10-perylene-tetracarboxylic acid,2,7-difluorofluorescein (Oregon Green™ 488-X), 5-carboxyfluorescein,Texas Red™-X, Alexa Fluor 430, 5-carboxytetramethylrhodamine (5-TAMRA),6-carboxytetramethylrhodamine (6-TAMRA), BODIPY FL, bimane, and AlexaFluor 350, 405, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647,660, 680, 700, and 750, and derivatives thereof, among many others. See,e.g., “The Handbook—A Guide to Fluorescent Probes and LabelingTechnologies,” Tenth Edition, available on the internet at probes (dot)invitrogen (dot) com/handbook. The fluorescent label can be one or moreof FAM, dRHO, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED,dROX, PET, BHQ, Gold540 and LIZ.

Using conventional techniques, an aptamer can be directly or indirectlylabeled. In a non-limiting example, the label is attached to the aptamerthrough biotin-streptavidin/avidin chemistry. For example, synthesize abiotinylated aptamer, which is then capable of binding a streptavidinmolecule that is itself conjugated to a detectable label; non-limitingexample is streptavidin, phycoerythrin conjugated (SAPE)). Methods forchemical coupling using multiple step procedures include biotinylation,coupling of trinitrophenol (TNP) or digoxigenin using for examplesuccinimide esters of these compounds. Biotinylation can be accomplishedby, for example, the use of D-biotinyl-N-hydroxysuccinimide Succinimidegroups react effectively with amino groups at pH values above 7, andpreferentially between about pH 8.0 and about pH 8.5. The labeling maycomprise a secondary labeling system. As a non-limiting example, theaptamer can be conjugated to biotin or digoxigenin. Target bound aptamercan be detected using streptavidin/avidin or anti-digoxigeninantibodies, respectively.

Various enzyme-substrate labels may also be used in conjunction with acomposition or method of the invention. Such enzyme-substrate labels areavailable commercially (e.g., U.S. Pat. No. 4,275,149). The enzymegenerally catalyzes a chemical alteration of a chromogenic substratethat can be measured using various techniques. For example, the enzymemay catalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Examples ofenzymatic labels include luciferases (e.g., firefly luciferase andbacterial luciferase; U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidasesuch as horseradish peroxidase (HRP), alkaline phosphatase (AP),β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Examples ofenzyme-substrate combinations include, but are not limited to,horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate,wherein the hydrogen peroxidase oxidizes a dye precursor (e.g.,orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethylbenzidinehydrochloride (TMB)); alkaline phosphatase (AP) with para-nitrophenylphosphate as chromogenic substrate; and β-D-galactosidase (β-D-Gal) witha chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) orfluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

Aptamer(s) can be linked to a substrate such as a planar substrate. Aplanar array generally contains addressable locations (e.g., pads,addresses, or micro-locations) of biomolecules in an array format. Thesize of the array will depend on the composition and end use of thearray. Arrays can be made containing from 2 different molecules to manythousands. Generally, the array comprises from two to as many as 100,000or more molecules, depending on the end use of the array and the methodof manufacture. A microarray for use with the invention comprises atleast one biomolecule that identifies or captures a biomarker present ina biosignature of interest, e.g., a cell, microRNA or other biomoleculeor vesicle that makes up the biosignature. In some arrays, multiplesubstrates are used, either of different or identical compositions.Accordingly, planar arrays may comprise a plurality of smallersubstrates.

The present invention can make use of many types of arrays for detectinga biomarker, e.g., a biomarker associated with a biosignature ofinterest. Useful arrays or microarrays include without limitation DNAmicroarrays, such as cDNA microarrays, oligonucleotide microarrays andSNP microarrays, microRNA arrays, protein microarrays, antibodymicroarrays, tissue microarrays, cellular microarrays (also calledtransfection microarrays), chemical compound microarrays, andcarbohydrate arrays (glycoarrays). These arrays are described in moredetail above. In some embodiments, microarrays comprise biochips thatprovide high-density immobilized arrays of recognition molecules (e.g.,aptamers or antibodies), where biomarker binding is monitored indirectly(e.g., via fluorescence).

An array or microarray that can be used to detect a biosignaturecomprising one or more aptamers of the invention can be made accordingto the methods described in U.S. Pat. Nos. 6,329,209; 6,365,418;6,406,921; 6,475,808; and 6,475,809, and U.S. patent application Ser.No. 10/884,269, each of which is herein incorporated by reference in itsentirety. Custom arrays to detect specific can be made using the methodsdescribed in these patents. Commercially available microarrays can alsobe used to carry out the methods of the invention, including withoutlimitation those from Affymetrix (Santa Clara, Calif.), Illumina (SanDiego, Calif.), Agilent (Santa Clara, Calif.), Exiqon (Denmark), orInvitrogen (Carlsbad, Calif.). Custom and/or commercial arrays includearrays for detection proteins, nucleic acids, and other biologicalmolecules and entities (e.g., cells, vesicles, virii) as describedherein.

In some embodiments, multiple capture molecules are disposed on anarray, e.g., proteins, peptides or additional nucleic acid molecules. Incertain embodiments, the proteins are immobilized using methods andmaterials that minimize the denaturing of the proteins, that minimizealterations in the activity of the proteins, or that minimizeinteractions between the protein and the surface on which they areimmobilized. The capture molecules can comprise one or more aptamer ofthe invention. In one embodiment, an array is constructed for thehybridization of a pool of aptamers. The array can then be used toidentify pool members that bind a sample, thereby facilitatingcharacterization of a phenotype. See FIGS. 10B-10C and relateddisclosure for further details.

Array surfaces useful may be of any desired shape, form, or size.Non-limiting examples of surfaces include chips, continuous surfaces,curved surfaces, flexible surfaces, films, plates, sheets, or tubes.Surfaces can have areas ranging from approximately a square micron toapproximately 500 cm². The area, length, and width of surfaces may bevaried according to the requirements of the assay to be performed.Considerations may include, for example, ease of handling, limitationsof the material(s) of which the surface is formed, requirements ofdetection systems, requirements of deposition systems (e.g., arrayers),or the like.

In certain embodiments, it is desirable to employ a physical means forseparating groups or arrays of binding islands or immobilizedbiomolecules: such physical separation facilitates exposure of differentgroups or arrays to different solutions of interest. Therefore, incertain embodiments, arrays are situated within microwell plates havingany number of wells. In such embodiments, the bottoms of the wells mayserve as surfaces for the formation of arrays, or arrays may be formedon other surfaces and then placed into wells. In certain embodiments,such as where a surface without wells is used, binding islands may beformed or molecules may be immobilized on a surface and a gasket havingholes spatially arranged so that they correspond to the islands orbiomolecules may be placed on the surface. Such a gasket is preferablyliquid tight. A gasket may be placed on a surface at any time during theprocess of making the array and may be removed if separation of groupsor arrays is no longer desired.

In some embodiments, the immobilized molecules can bind to one or morebiomarkers present in a biological sample contacting the immobilizedmolecules. Contacting the sample typically comprises overlaying thesample upon the array.

Modifications or binding of molecules in solution or immobilized on anarray can be detected using detection techniques known in the art.Examples of such techniques include immunological techniques such ascompetitive binding assays and sandwich assays; fluorescence detectionusing instruments such as confocal scanners, confocal microscopes, orCCD-based systems and techniques such as fluorescence, fluorescencepolarization (FP), fluorescence resonant energy transfer (FRET), totalinternal reflection fluorescence (TIRF), fluorescence correlationspectroscopy (FCS); colorimetric/spectrometric techniques; surfaceplasmon resonance, by which changes in mass of materials adsorbed atsurfaces are measured; techniques using radioisotopes, includingconventional radioisotope binding and scintillation proximity assays(SPA); mass spectroscopy, such as matrix-assisted laserdesorption/ionization mass spectroscopy (MALDI) and MALDI-time of flight(TOF) mass spectroscopy; ellipsometry, which is an optical method ofmeasuring thickness of protein films; quartz crystal microbalance (QCM),a very sensitive method for measuring mass of materials adsorbing tosurfaces; scanning probe microscopies, such as atomic force microscopy(AFM), scanning force microscopy (SFM) or scanning electron microscopy(SEM); and techniques such as electrochemical, impedance, acoustic,microwave, and IR/Raman detection. See, e.g., Mere L, et al.,“Miniaturized FRET assays and microfluidics: key components forultra-high-throughput screening,” Drug Discovery Today 4(8):363-369(1999), and references cited therein; Lakowicz J R, Principles ofFluorescence Spectroscopy, 2nd Edition, Plenum Press (1999), or Jain KK: Integrative Omics, Pharmacoproteomics, and Human Body Fluids. In:Thongboonkerd V, ed., ed. Proteomics of Human Body Fluids: Principles,Methods and Applications. Volume 1: Totowa, N.J.: Humana Press, 2007,each of which is herein incorporated by reference in its entirety.

Microarray technology can be combined with mass spectroscopy (MS)analysis and other tools. Electrospray interface to a mass spectrometercan be integrated with a capillary in a microfluidics device. Forexample, one commercially available system contains eTag reporters thatare fluorescent labels with unique and well-defined electrophoreticmobilities; each label is coupled to biological or chemical probes viacleavable linkages. The distinct mobility address of each eTag reporterallows mixtures of these tags to be rapidly deconvoluted and quantitatedby capillary electrophoresis. This system allows concurrent geneexpression, protein expression, and protein function analyses from thesame sample Jain K K: Integrative Omics, Pharmacoproteomics, and HumanBody Fluids. In: Thongboonkerd V, ed., ed. Proteomics of Human BodyFluids: Principles, Methods and Applications. Volume 1: Totowa, N.J.:Humana Press, 2007, which is herein incorporated by reference in itsentirety.

A biochip can include components for a microfluidic or nanofluidicassay. A microfluidic device can be used for isolating or analyzingbiomarkers, such as determining a biosignature. Microfluidic systemsallow for the miniaturization and compartmentalization of one or moreprocesses for detecting a biosignature, and other processes. Themicrofluidic devices can use one or more detection reagents in at leastone aspect of the system, and such a detection reagent can be used todetect one or more biomarkers. Various probes, antibodies, proteins, orother binding agents can be used to detect a biomarker within themicrofluidic system. The detection agents, e.g., oligonucleotide probesof the invention, may be immobilized in different compartments of themicrofluidic device or be entered into a hybridization or detectionreaction through various channels of the device.

Nanofabrication techniques are opening up the possibilities forbiosensing applications that rely on fabrication of high-density,precision arrays, e.g., nucleotide-based chips and protein arraysotherwise known as heterogeneous nanoarrays. Nanofluidics allows afurther reduction in the quantity of fluid analyte in a microchip tonanoliter levels, and the chips used here are referred to as nanochips.See, e.g., Unger M et al., Biotechniques 1999; 27(5):1008-14, Kartalov EP et al., Biotechniques 2006; 40(1):85-90, each of which are hereinincorporated by reference in their entireties. Commercially availablenanochips currently provide simple one step assays such as totalcholesterol, total protein or glucose assays that can be run bycombining sample and reagents, mixing and monitoring of the reaction.Gel-free analytical approaches based on liquid chromatography (LC) andnanoLC separations (Cutillas et al. Proteomics, 2005; 5: 101-112 andCutillas et al., Mol Cell Proteomics 2005; 4: 1038-1051, each of whichis herein incorporated by reference in its entirety) can be used incombination with the nanochips.

An array suitable for identifying a disease, condition, syndrome orphysiological status can be included in a kit. A kit can include, anaptamer of the invention, including as non-limiting examples, one ormore reagents useful for preparing molecules for immobilization ontobinding islands or areas of an array, reagents useful for detectingbinding of biomarkers to immobilized molecules, e.g., aptamers, andinstructions for use.

Further provided herein is a rapid detection device that facilitates thedetection of a particular biosignature in a biological sample. Thedevice can integrate biological sample preparation with polymerase chainreaction (PCR) on a chip. The device can facilitate the detection of aparticular biosignature of a vesicle in a biological sample, and anexample is provided as described in Pipper et al., Angewandte Chemie,47(21), p. 3900-3904 (2008), which is herein incorporated by referencein its entirety. A biosignature can be incorporated usingmicro-/nano-electrochemical system (MEMS/NEMS) sensors and oral fluidfor diagnostic applications as described in Li et al., Adv Dent Res18(1): 3-5 (2005), which is herein incorporated by reference in itsentirety.

As an alternative to planar arrays, assays using particles, such as beadbased assays are also capable of use with an aptamer of the invention.Aptamers are easily conjugated with commercially available beads. See,e.g., Srinivas et al. Anal. Chem. 2011 Oct. 21, Aptamer functionalizedMicrogel Particles for Protein Detection; See also, review article onaptamers as therapeutic and diagnostic agents, Brody and Gold, Rev. Mol.Biotech. 2000, 74:5-13.

Multiparametric assays or other high throughput detection assays usingbead coatings with cognate ligands and reporter molecules with specificactivities consistent with high sensitivity automation can be used. In abead based assay system, a binding agent such as an antibody or aptamercan be immobilized on an addressable microsphere. Each binding agent foreach individual binding assay can be coupled to a distinct type ofmicrosphere (i.e., microbead) and the assay reaction takes place on thesurface of the microsphere, such as depicted in FIG. 1B. In anon-limiting example, a binding agent for a cell or microvesicle can bea capture antibody or aptamer coupled to a bead. Dyed microspheres withdiscrete fluorescence intensities are loaded separately with theirappropriate binding agent or capture probes. The different bead setscarrying different binding agents can be pooled as desired to generatecustom bead arrays. Bead arrays are then incubated with the sample in asingle reaction vessel to perform the assay.

Bead-based assays can be used with one or more aptamers of theinvention. A bead substrate can provide a platform for attaching one ormore binding agents, including aptamer(s). For multiplexing, multipledifferent bead sets (e.g., Illumina, Luminex) can have different bindingagents (specific to different target molecules). For example, a bead canbe conjugated to an aptamer of the invention used to detect the presence(quantitatively or qualitatively) of an antigen of interest, or it canalso be used to isolate a component present in a selected biologicalsample (e.g., cell, cell-fragment or vesicle comprising the targetmolecule to which the aptamer is configured to bind or associate). Anymolecule of organic origin can be successfully conjugated to apolystyrene bead through use of commercially available kits.

One or more aptamers of the invention can be used with any bead basedsubstrate, including but not limited to magnetic capture method,fluorescence activated cell sorting (FACS) or laser cytometry. Magneticcapture methods can include, but are not limited to, the use ofmagnetically activated cell sorter (MACS) microbeads or magnetic columnsExamples of bead or particle based methods that can be modified to usean aptamer of the invention include methods and bead systems describedin U.S. Pat. Nos. 4,551,435, 4,795,698, 4,925,788, 5,108,933, 5,186,827,5,200,084 or 5,158,871; 7,399,632; 8,124,015; 8,008,019; 7,955,802;7,445,844; 7,274,316; 6,773,812; 6,623,526; 6,599,331; 6,057,107;5,736,330; International Patent Publication No. WO/2012/174282;WO/1993/022684.

Isolation or detection of circulating biomarkers, e.g., proteinantigens, from a biological sample, or of the biomarker-comprisingcells, cell fragments or vesicles may also be achieved using an aptamerof the invention in a cytometry process. As a non-limiting example,aptamers of the invention can be used in an assay comprising using aparticle such as a bead or microsphere. The invention provides aptamersas binding agents, which may be conjugated to the particle. Flowcytometry can be used for sorting microscopic particles suspended in astream of fluid. As particles pass through they can be selectivelycharged and on their exit can be deflected into separate paths of flow.It is therefore possible to separate populations from an original mix,such as a biological sample, with a high degree of accuracy and speed.Flow cytometry allows simultaneous multiparametric analysis of thephysical and/or chemical characteristics of single cells flowing throughan optical/electronic detection apparatus. A beam of light, usuallylaser light, of a single frequency (color) is directed onto ahydrodynamically focused stream of fluid. A number of detectors areaimed at the point where the stream passes through the light beam; onein line with the light beam (Forward Scatter or FSC) and severalperpendicular to it (Side Scatter or SSC) and one or more fluorescentdetectors.

Each suspended particle passing through the beam scatters the light insome way, and fluorescent chemicals in the particle may be excited intoemitting light at a lower frequency than the light source. Thiscombination of scattered and fluorescent light is picked up by thedetectors, and by analyzing fluctuations in brightness at each detector(one for each fluorescent emission peak), it is possible to deducevarious facts about the physical and chemical structure of eachindividual particle. FSC correlates with the cell size and SSC dependson the inner complexity of the particle, such as shape of the nucleus,the amount and type of cytoplasmic granules or the membrane roughness.Some flow cytometers have eliminated the need for fluorescence and useonly light scatter for measurement.

Flow cytometers can analyze several thousand particles every second in“real time” and can actively separate out and isolate particles havingspecified properties. They offer high-throughput automatedquantification, and separation, of the set parameters for a high numberof single cells during each analysis session. Flow cytometers can havemultiple lasers and fluorescence detectors, allowing multiple labels tobe used to more precisely specify a target population by theirphenotype. Thus, a flow cytometer, such as a multicolor flow cytometer,can be used to detect targets of interest using multiple fluorescentlabels or colors. In some embodiments, the flow cytometer can also sortor isolate different targets of interest, such as by size or bydifferent markers.

The flow cytometer may have one or more lasers, such as 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more lasers. In some embodiments, the flow cytometercan detect more than one color or fluorescent label, such as at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20different colors or fluorescent labels. For example, the flow cytometercan have at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 fluorescence detectors.

Examples of commercially available flow cytometers include, but are notlimited to the MoFlo™ XDP Cell Sorter (Beckman Coulter, Brea, Calif.),MoFlo™ Legacy Cell Sorter (Beckman Coulter, Brea, Calif.), BD FACSAria™Cell Sorter (BD Biosciences, San Jose, Calif.), BD™ LSRII (BDBiosciences, San Jose, Calif.), and BD FACSCalibur™ (BD Biosciences, SanJose, Calif.). Use of multicolor or multi-fluor cytometers can be usedin multiplex analysis. In some embodiments, the flow cytometer can sort,and thereby collect or sort more than one population of cells,microvesicles, or particles, based one or more characteristics. Forexample, two populations differ in size, such that the populations havea similar size range can be differentially detected or sorted. Inanother embodiment, two different populations are differentiallylabeled.

The data resulting from flow-cytometers can be plotted in 1 dimension toproduce histograms or seen in 2 dimensions as dot plots or in 3dimensions with newer software. The regions on these plots can besequentially separated by a series of subset extractions which aretermed gates. Specific gating protocols exist for diagnostic andclinical purposes especially in relation to hematology. The plots areoften made on logarithmic scales. Because different fluorescent dye'semission spectra overlap, signals at the detectors have to becompensated electronically as well as computationally. Fluorophores forlabeling biomarkers may include those described in Ormerod, FlowCytometry 2nd ed., Springer-Verlag, New York (1999), and in Nida et al.,Gynecologic Oncology 2005; 4 889-894 which is incorporated herein byreference. In a multiplexed assay, including but not limited to a flowcytometry assay, one or more different target molecules can be assessedusing an aptamer of the invention.

One or more aptamer of the invention can be disposed on any usefulplanar or bead substrate. In one aspect of the invention one or moreaptamer of the invention is disposed on a microfluidic device, therebyfacilitating assessing, characterizing or isolating a component of abiological sample comprising a polypeptide antigen of interest or afunctional fragment thereof. For example, the circulating antigen or acell, cell fragment or cell-derived microvesicles comprising the antigencan be assessed using one or more aptamers of the invention(alternatively along with additional binding agents). Microfluidicdevices, which may also be referred to as “lab-on-a-chip” systems,biomedical micro-electro-mechanical systems (bioMEMs), or multicomponentintegrated systems, can be used for isolating and analyzing suchentities. Such systems miniaturize and compartmentalize processes thatallow for detection of biosignatures and other processes.

A microfluidic device can also be used for isolation of a cell, cellfragment or cell-derived microvesicles through size differential oraffinity selection. For example, a microfluidic device can use one morechannels for isolating entities from a biological sample based on sizeor by using one or more binding agents. A biological sample can beintroduced into one or more microfluidic channels, which selectivelyallows the passage of the entity. The selection can be based on aproperty such as the size, shape, deformability, or biosignature.

In one embodiment, a heterogeneous population of cells, cell fragments,microvesicles or other biomarkers (e.g., protein complexes) isintroduced into a microfluidic device, and one or more differenthomogeneous populations of such entities can be obtained. For example,different channels can have different size selections or binding agentsto select for different populations of such entities. Thus, amicrofluidic device can isolate a plurality of entities wherein at leasta subset of the plurality comprises a different biosignature fromanother subset of the plurality. For example, the microfluidic devicecan isolate at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50,60, 70, 80, 90, or 100 different subsets, wherein each subset comprisesa different biosignature.

In some embodiments, the microfluidic device can comprise one or morechannels that permit further enrichment or selection of targets ofinterest. A population that has been enriched after passage through afirst channel can be introduced into a second channel, which allows thepassage of the desired population to be further enriched, such asthrough one or more binding agents present in the second channel.

Array-based assays and bead-based assays can be used with a microfluidicdevice. For example, the binding agent, such as an oligonucleotideprobe, can be coupled to beads and the binding reaction between thebeads and targets of the binding agent can be performed in amicrofluidic device. Multiplexing can also be performed using amicrofluidic device. Different compartments can comprise differentbinding agents for different target populations. In one embodiment, eachpopulation has a different biosignature. The hybridization reactionbetween the microsphere and target can be performed in a microfluidicdevice and the reaction mixture can be delivered to a detection device.The detection device, such as a dual or multiple laser detection systemcan be part of the microfluidic system and can use a laser to identifyeach bead or microsphere by its color-coding, and another laser candetect the hybridization signal associated with each bead.

Any appropriate microfluidic device can be used in the methods of theinvention. Examples of microfluidic devices that may be used include butare not limited to those described in U.S. Pat. Nos. 7,591,936,7,581,429, 7,579,136, 7,575,722, 7,568,399, 7,552,741, 7,544,506,7,541,578, 7,518,726, 7,488,596, 7,485,214, 7,467,928, 7,452,713,7,452,509, 7,449,096, 7,431,887, 7,422,725, 7,422,669, 7,419,822,7,419,639, 7,413,709, 7,411,184, 7,402,229, 7,390,463, 7,381,471,7,357,864, 7,351,592, 7,351,380, 7,338,637, 7,329,391, 7,323,140,7,261,824, 7,258,837, 7,253,003, 7,238,324, 7,238,255, 7,233,865,7,229,538, 7,201,881, 7,195,986, 7,189,581, 7,189,580, 7,189,368,7,141,978, 7,138,062, 7,135,147, 7,125,711, 7,118,910, 7,118,661,7,640,947, 7,666,361, 7,704,735; and International Patent Publication WO2010/072410; each of which patents or applications are incorporatedherein by reference in their entirety. Another example for use withmethods disclosed herein is described in Chen et al., “Microfludicisolation and transcriptome analysis of serum vesicles,” Lab on a Chip,Dec. 8, 2009 DOI: 10.1039/b916199f.

Other microfluidic devices for use with the invention include devicescomprising elastomeric layers, valves and pumps, including withoutlimitation those disclosed in U.S. Pat. Nos. 5,376,252, 6,408,878,6,645,432, 6,719,868, 6,793,753, 6,899,137, 6,929,030, 7,040,338,7,118,910, 7,144,616, 7,216,671, 7,250,128, 7,494,555, 7,501,245,7,601,270, 7,691,333, 7,754,010, 7,837,946; U.S. Patent Application Nos.2003/0061687, 2005/0084421, 2005/0112882, 2005/0129581, 2005/0145496,2005/0201901, 2005/0214173, 2005/0252773, 2006/0006067; and EP PatentNos. 0527905 and 1065378; each of which application is hereinincorporated by reference.

The microfluidic device can have one or more binding agents attached toa surface in a channel, or present in a channel. For example, themicrochannel can have one or more capture agents, such as anoligonucleotide probe of the invention. The surface of the channel canalso be contacted with a blocking aptamer if desired. In one embodiment,a microchannel surface is treated with avidin/streptavidin and a captureagent, such as an antibody or aptamer, that is biotinylated can beinjected into the channel to bind the avidin. In other embodiments, thecapture agents are present in chambers or other components of amicrofluidic device. The capture agents can also be attached to beadsthat can be manipulated to move through the microfluidic channels. Inone embodiment, the capture agents are attached to magnetic beads. Thebeads can be manipulated using magnets.

A biological sample can be flowed into the microfluidic device, or amicrochannel, at rates such as at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 μlper minute, such as between about 1-50, 5-40, 5-30, 3-20 or 5-15 μl perminute. One or more targets of interest can be captured and directlydetected in the microfluidic device. Alternatively, the captured targetmay be released and exit the microfluidic device prior to analysis. Inanother embodiment, one or more captured cells or microvesicles arelysed in the microchannel and the lysate can be analyzed. Lysis buffercan be flowed through the channel. The lysate can be collected andanalyzed, such as performing RT-PCR, PCR, mass spectrometry, Westernblotting, or other assays, to detect one or more biomarkers of thecaptured cells or microvesicles.

Microvesicles and related biomarkers can be analyzed using theoligonucleotide probes of the invention. Microvesicle isolation can beperformed using various techniques as, including without limitation sizeexclusion chromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, affinity capture, immunoassay,immunoprecipitation, microfluidic separation, flow cytometry, polymericisolation (e.g., using polyethylene glycol (PEG)) or combinationsthereof. Methods and techniques for microvesicle and vesicular payloadisolation and analysis are disclosed in International Patent ApplicationNos. PCT/US2009/62880, filed Oct. 30, 2009; PCT/US2009/006095, filedNov. 12, 2009; PCT/US2011/26750, filed Mar. 1, 2011; PCT/US2011/031479,filed Apr. 6, 2011; PCT/US11/48327, filed Aug. 18, 2011;PCT/US2008/71235, filed Jul. 25, 2008; PCT/US10/58461, filed Nov. 30,2010; PCT/US2011/21160, filed Jan. 13, 2011; PCT/US2013/030302, filedMar. 11, 2013; PCT/US12/25741, filed Feb. 17, 2012; PCT/2008/76109,filed Sep. 12, 2008; PCT/US12/42519, filed Jun. 14, 2012;PCT/US12/50030, filed Aug. 8, 2012; PCT/US12/49615, filed Aug. 3, 2012;PCT/US12/41387, filed Jun. 7, 2012; PCT/US2013/072019, filed Nov. 26,2013; PCT/US2014/039858, filed May 28, 2013; PCT/IB2013/003092, filedOct. 23, 2013; PCT/US13/76611, filed Dec. 19, 2013; PCT/US14/53306,filed Aug. 28, 2014; and PCT/US15/62184, filed Nov. 23, 2015;PCT/US16/40157, filed Jun. 29, 2016; PCT/US16/44595, filed Jul. 28,2016; and PCT/US16/21632, filed Mar. 9, 2016; each of which applicationsis incorporated herein by reference in its entirety.

The compositions and methods of the invention can be used in and withvarious immune assay formats Immunoaffinity assays can be based onantibodies and aptamers selectively immunoreactive with proteins orother biomarkers of interest. These techniques include withoutlimitation immunoprecipitation, Western blot analysis, molecular bindingassays, enzyme-linked immunosorbent assay (ELISA), enzyme-linkedimmunofiltration assay (ELIFA), fluorescence activated cell sorting(FACS), immunohistochemistry (IHC) and the like. For example, anoptional method of detecting the expression of a biomarker in a samplecomprises contacting the sample with an antibody or aptamer against thebiomarker, or an immunoreactive fragment thereof, or a recombinantprotein containing an antigen binding region against the biomarker; andthen detecting the binding of the biomarker in the sample. Variousmethods for producing antibodies and aptamers are known in the art. Suchbinding agents can be used to immunoprecipitate specific proteins fromsolution samples or to immunoblot proteins separated by, e.g.,polyacrylamide gels Immunocytochemical methods can also be used indetecting specific protein polymorphisms in tissues or cells. Otherwell-known immunoassay techniques can also be used including, e.g.,ELISA, radioimmunoassay (RIA), immunoradiometric assays (IRMA) andimmunoenzymatic assays (IEMA), including sandwich assays. See, e.g.,U.S. Pat. Nos. 4,376,110 and 4,486,530, both of which are incorporatedherein by reference.

In alternative methods, a sample may be contacted with an antibody oraptamer specific for a biomarker under conditions sufficient for acomplex to form, and then detecting such complex. The presence of thebiomarker may be detected in a number of ways, such as by Westernblotting and ELISA procedures for assaying a wide variety of tissues andsamples, including bodily fluids such as plasma or serum. A wide rangeof immunoassay techniques using such an assay format are available, see,e.g., U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These includeboth single-site and two-site or “sandwich” assays of thenon-competitive types, as well as in the traditional competitive bindingassays. These assays also include direct binding of a labelled antibodyor aptamer to a target biomarker.

There are a number of variations of the sandwich assay technique whichcan be encompassed within the present invention. In a typical forwardassay, an unlabeled binding agent, e.g., an antibody or aptamer, isimmobilized on a solid substrate, and the sample to be tested broughtinto contact with the bound molecule. After a suitable period of timesufficient to allow formation of an complex, a second binding agentspecific to the antigen, labelled with a reporter molecule capable ofproducing a detectable signal is then added and incubated, allowing timesufficient for the formation of another complex comprising the labelledbinding agent. Any unreacted material is washed away, and the presenceof the antigen is determined by observation of a signal produced by thereporter molecule. The results may either be qualitative, by simpleobservation of the visible signal, or may be quantitated by comparingwith a control sample containing known amounts of biomarker.

Variations on the above assay include a simultaneous assay, in whichboth sample and labelled binding agent are added simultaneously to thetethered binding agent. In a typical forward sandwich assay, a firstbinding agent, e.g., an antibody or aptamer, having specificity for atissue/cell/biomarker or such target of interest is either covalently orpassively bound to a solid surface. The solid surface is typically glassor a polymer, the most commonly used polymers being cellulose,polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.The solid supports may be in the form of tubes, beads, discs ofmicroplates, or any other surface suitable for conducting animmunoassay. The binding processes generally consist of cross-linking,covalently binding or physically adsorbing, the polymer-antibody complexto the support, which is then washed in preparation for the test sample.An aliquot of the sample to be tested is then added to the solid phasecomplex and incubated for a period of time sufficient (e.g., 2-40minutes or overnight) and under suitable conditions (e.g., from roomtemperature to 40° C. such as between 25° C. and 32° C. inclusive) toallow binding of the target to the support. Following the incubationperiod, the support is washed and incubated with a second binding agentspecific for a portion of the biomarker. The second binding agent islinked to a reporter molecule which is used to indicate the binding ofthe second binding agent to the molecular marker.

An alternative method involves immobilizing the target biomarkers in thesample and then exposing the immobilized target to specific bindingagents, e.g., antibodies or aptamers, which may or may not be labelledwith a reporter molecule. Depending on the amount of target and thestrength of the reporter molecule signal, a bound target may bedetectable by direct labelling with the binding agent. Alternatively, asecond labelled binding agent, specific to the first binding agent, isexposed to the first target complex to form a tertiary complex. Thecomplex is detected by the signal emitted by the reporter molecule. A“reporter molecule” includes molecule which, by its chemical nature,provides an analytically identifiable signal which allows the detectionof antigen-bound complexes. Some commonly used reporter molecules inthis type of assay include enzymes, fluorophores or radionuclidecontaining molecules (i.e. radioisotopes) and chemiluminescentmolecules. Examples of such detectable labels are disclosed herein.

In the case of an enzyme immunoassay, an enzyme is conjugated to thesecondary binding agent. Commonly used enzymes include horseradishperoxidase, glucose oxidase, β-galactosidase and alkaline phosphatase,amongst others. The substrates to be used with the specific enzymes aregenerally chosen for the production, upon hydrolysis by thecorresponding enzyme, of a detectable color change. Examples of suitableenzymes include alkaline phosphatase and peroxidase. It is also possibleto employ fluorogenic substrates, which yield a fluorescent productrather than the chromogenic substrates noted above. In all cases, theenzyme-labelled binding agent is added to the first bound molecularmarker complex, allowed to bind, and then the excess reagent is washedaway. A solution containing the appropriate substrate is then added tothe tertiary complex comprising primary binding agent, antigen, andsecondary binding agent. The substrate will react with the enzyme linkedto the secondary binding agent, giving a qualitative visual signal,which may be further quantitated, usually spectrophotometrically, togive an indication of the amount of antigen which was present in thesample. Alternately, fluorescent compounds, such as fluorescein andrhodamine, may be chemically coupled to secondary binding agent withoutaltering their binding capacity. When activated by illumination withlight of a particular wavelength, the fluorochrome-labelled secondarybinding agent adsorbs the light energy, inducing a state to excitabilityin the molecule, followed by emission of the light at a characteristiccolor visually detectable with a light microscope. As in the EIA, thefluorescent labelled secondary binding agent is allowed to bind toantigen complex. After washing off the unbound reagent, the remainingtertiary complex is then exposed to the light of the appropriatewavelength. The fluorescence observed indicates the presence of themolecular marker of interest. Immunofluorescence and EIA techniques areboth very well established in the art. However, other reportermolecules, such as radioisotope, chemiluminescent or bioluminescentmolecules, may also be employed.

Immunohistochemistry (IHC) is a process of localizing antigens (e.g.,proteins) in cells of a tissue using binding agents (e.g., antibodies oraptamers) specifically to antigens in the tissues. The antigen-bindingbinding agent can be conjugated or fused to a tag that allows itsdetection, e.g., via visualization. In some embodiments, the tag is anenzyme that can catalyze a color-producing reaction, such as alkalinephosphatase or horseradish peroxidase. The enzyme can be fused to thebinding agent or non-covalently bound, e.g., using abiotin-avadin/streptavidin system. Alternatively, the binding agent canbe tagged with a fluorophore, such as fluorescein, rhodamine, DyLightFluor or Alexa Fluor. The binding agent can be directly tagged or it canitself be recognized by a secondary detection binding agent (antibody orantigen) that carries the tag. Using IHC, one or more proteins may bedetected. The expression of a gene product can be related to itsstaining intensity compared to control levels. In some embodiments, thegene product is considered differentially expressed if its stainingvaries at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5,2.7, 3.0, 4, 5, 6, 7, 8, 9 or 10-fold in the sample versus the control.

IHC comprises the application of such immunoassay formats tohistochemical techniques. In an illustrative example, a tissue sectionis mounted on a slide and is incubated with a binding agent. The bindingagents are typically polyclonal or monoclonal antibodies, and can beaptamers such as oligonucleotide probes of the invention, specific tothe antigen. The primary reaction comprises contacting the tissuesection with this primary binding agent, forming primary complexes. Theantigen-antibody signal is then amplified using a second binding agentconjugated to a complex of that can provide a visible signal, such asenzymes including without limitation peroxidase antiperoxidase (PAP),avidin-biotin-peroxidase (ABC) or avidin-biotin alkaline phosphatase. Inthe presence of substrate and chromogen, the enzyme forms a coloreddeposit at the sites of primary complexes Immunofluorescence is analternate approach to visualize antigens. In this technique, the primarysignal is amplified using a second binding agent conjugated to afluorochrome. On UV light absorption, the fluorochrome emits its ownlight at a longer wavelength (fluorescence), thus allowing localizationof the primary complexes.

The invention provides methods of performing an IHC assay using anoligonucleotide probe library. This may be referred to as a polyligandhistochemistry assay (PHC). As an example of this approach, a tissuesection is contacted with an enriched oligonucleotide probe library.Members of the library can be labeled, e.g., with a biotin molecule,digoxigenin, or other label as appropriate. The bound library membersare visualized using a secondary labeling system, e.g.,streptavidin-horse radish peroxidase (SA-HRP) or anti-digoxigenin horseradish peroxidase. The resulting slides can be read and scored as intypical antibody based IHC methods. See Examples 19-31 herein.

Oligonucleotide Probes/Aptamers

Aptamers have a number of desirable characteristics for use astherapeutics and diagnostics including high specificity and affinity,biological efficacy, and excellent pharmacokinetic properties. Inaddition, they offer certain advantages over antibodies and otherprotein biologics. For example, aptamers are produced by an entirely invitro process, allowing for the rapid synthesis. In vitro selectionallows the specificity and affinity of the aptamer to be tightlycontrolled. In addition, aptamers as a class have demonstrated little orno toxicity or immunogenicity. Whereas the efficacy of many monoclonalantibodies can be severely limited by immune response to antibodiesthemselves, it is difficult to elicit antibodies to aptamers most likelybecause aptamers cannot be presented by T-cells via the MHC and theimmune response is generally trained not to recognize nucleic acidfragments. Whereas most currently approved antibody therapeutics areadministered by intravenous infusion (typically over 2-4 hours),aptamers can be administered by subcutaneous injection. This differenceis primarily due to the comparatively low solubility and thus largevolumes necessary for most therapeutic mAbs. With good solubility (>150mg/mL) and comparatively low molecular weight (aptamer: 10-50 kDa;antibody: 150 kDa), a weekly dose of aptamer may be delivered byinjection in a volume of less than 0.5 mL. In addition, the small sizeof aptamers allows them to penetrate into areas of conformationalconstrictions that do not allow for antibodies or antibody fragments topenetrate, presenting yet another advantage of aptamer-basedtherapeutics or prophylaxis.

Aptamers are chemically synthesized and are readily scaled as needed tomeet production demand for diagnostic or therapeutic applications. Inaddition, aptamers are chemically robust. They can be adapted to regainactivity following exposure to factors such as heat and denaturants andcan be stored for extended periods (>1 yr) at room temperature aslyophilized powders.

SELEX

The classical method for generating an aptamer is with the processentitled “Systematic Evolution of Ligands by Exponential Enrichment”(“SELEX”) generally described in, e.g., U.S. patent application Ser. No.07/536,428, filed Jun. 11, 1990, now abandoned, U.S. Pat. No. 5,475,096entitled “Nucleic Acid Ligands”, and U.S. Pat. No. 5,270,163 (see alsoWO 91/19813) entitled “Nucleic Acid Ligands.” Each SELEX-identifiednucleic acid ligand, i.e., each aptamer (or oligonucleotide probe), is aspecific ligand of a given target compound or molecule. The SELEXprocess is based on the insight that nucleic acids have sufficientcapacity for forming a variety of two- and three-dimensional structuresand sufficient chemical versatility available within their monomers toact as ligands (i.e., form specific binding pairs) with any variety ofchemical compounds, whether monomeric or polymeric. Molecules of anysize or composition can serve as targets.

SELEX relies as a starting point upon a large library or pool of singlestranded oligonucleotides comprising randomized sequences. Theoligonucleotides can be modified or unmodified DNA, RNA, or DNA/RNAhybrids. In some examples, the pool comprises 100% random or partiallyrandom oligonucleotides. In other examples, the pool comprises random orpartially random oligonucleotides containing at least one fixed and/orconserved sequence incorporated within randomized sequence. In otherexamples, the pool comprises random or partially random oligonucleotidescontaining at least one fixed and/or conserved sequence at its 5′ and/or3′ end which may comprise a sequence shared by all the molecules of theoligonucleotide pool. Fixed sequences are sequences such ashybridization sites for PCR primers, promoter sequences for RNApolymerases (e.g., T3, T4, T7, and SP6), restriction sites, orhomopolymeric sequences, such as poly A or poly T tracts, catalyticcores, sites for selective binding to affinity columns, and othersequences to facilitate cloning and/or sequencing of an oligonucleotideof interest. Conserved sequences are sequences, other than thepreviously described fixed sequences, shared by a number of aptamersthat bind to the same target.

The oligonucleotides of the pool preferably include a randomizedsequence portion as well as fixed sequences necessary for efficientamplification. Typically the oligonucleotides of the starting poolcontain fixed 5′ and 3′ terminal sequences which flank an internalregion of 30-50 random nucleotides. The randomized nucleotides can beproduced in a number of ways including chemical synthesis and sizeselection from randomly cleaved cellular nucleic acids. Sequencevariation in test nucleic acids can also be introduced or increased bymutagenesis before or during the selection/amplification iterations.

The random sequence portion of the oligonucleotide can be of anyappropriate length and can comprise ribonucleotides and/ordeoxyribonucleotides and can include modified or non-natural nucleotidesor nucleotide analogs. See, e.g. U.S. Pat. Nos. 5,958,691; 5,660,985;5,958,691; 5,698,687; 5,817,635; 5,672,695, and PCT Publication WO92/07065. Random oligonucleotides can be synthesized fromphosphodiester-linked nucleotides using solid phase oligonucleotidesynthesis techniques well known in the art. See, e.g., Froehler et al.,Nucl. Acid Res. 14:5399-5467 (1986) and Froehler et al., Tet. Lett.27:5575-5578 (1986). Random oligonucleotides can also be synthesizedusing solution phase methods such as triester synthesis methods. See,e.g., Sood et al., Nucl. Acid Res. 4:2557 (1977) and Hirose et al., Tet.Lett., 28:2449 (1978). Typical syntheses carried out on automated DNAsynthesis equipment yield 10¹⁴-10¹⁶ individual molecules, a numbersufficient for most SELEX experiments. Sufficiently large regions ofrandom sequence in the sequence design increases the likelihood thateach synthesized molecule is likely to represent a unique sequence.

The starting library of oligonucleotides may be generated by automatedchemical synthesis on a DNA synthesizer. To synthesize randomizedsequences, mixtures of all four nucleotides are added at each nucleotideaddition step during the synthesis process, allowing for randomincorporation of nucleotides. As stated above, in one embodiment, randomoligonucleotides comprise entirely random sequences; however, in otherembodiments, random oligonucleotides can comprise stretches of nonrandomor partially random sequences. Partially random sequences can be createdby adding the four nucleotides in different molar ratios at eachaddition step.

The starting library of oligonucleotides may be for example, RNA, DNA,or RNA/DNA hybrid. A starting RNA library can be generated bytranscribing a DNA library in vitro using T7 RNA polymerase or modifiedT7 RNA polymerases and purified. The library is then mixed with thetarget under conditions favorable for binding and subjected to step-wiseiterations of binding, partitioning and amplification, using the samegeneral selection scheme, to achieve virtually any desired criterion ofbinding affinity and selectivity. More specifically, starting with amixture containing the starting pool of nucleic acids, the SELEX methodincludes steps of: (a) contacting the mixture with the target underconditions favorable for binding; (b) partitioning unbound nucleic acidsfrom those nucleic acids which have bound specifically to targetmolecules; (c) dissociating the nucleic acid-target complexes; (d)amplifying the nucleic acids dissociated from the nucleic acid-targetcomplexes to yield a ligand-enriched mixture of nucleic acids; and (e)reiterating the steps of binding, partitioning, dissociating andamplifying through as many cycles as desired to yield highly specific,high affinity nucleic acid ligands to the target molecule. In thoseinstances where RNA aptamers are being selected, the SELEX methodfurther comprises the steps of: (i) reverse transcribing the nucleicacids dissociated from the nucleic acid-target complexes beforeamplification in step (d); and (ii) transcribing the amplified nucleicacids from step (d) before restarting the process.

Within a nucleic acid mixture containing a large number of possiblesequences and structures, there is a wide range of binding affinitiesfor a given target. A nucleic acid mixture comprising, for example, a 20nucleotide randomized segment can have 4²⁰ candidate possibilities.Those which have the higher affinity constants for the target are mostlikely to bind to the target. After partitioning, dissociation andamplification, a second nucleic acid mixture is generated, enriched forthe higher binding affinity candidates. Additional rounds of selectionprogressively favor better ligands until the resulting nucleic acidmixture is predominantly composed of only one or a few sequences. Thesecan then be cloned, sequenced and individually tested for bindingaffinity as pure ligands or aptamers.

Cycles of selection and amplification are repeated until a desired goalis achieved. In the most general case, selection/amplification iscontinued until no significant improvement in binding strength isachieved on repetition of the cycle. The method is typically used tosample approximately 10¹⁴ different nucleic acid species but may be usedto sample as many as about 10¹⁸ different nucleic acid species.Generally, nucleic acid aptamer molecules are selected in a 5 to 20cycle procedure. In one embodiment, heterogeneity is introduced only inthe initial selection stages and does not occur throughout thereplicating process.

In one embodiment of SELEX, the selection process is so efficient atisolating those nucleic acid ligands that bind most strongly to theselected target, that only one cycle of selection and amplification isrequired. Such an efficient selection may occur, for example, in achromatographic-type process wherein the ability of nucleic acids toassociate with targets bound on a column operates in such a manner thatthe column is sufficiently able to allow separation and isolation of thehighest affinity nucleic acid ligands.

In many cases, it is not necessarily desirable to perform the iterativesteps of SELEX until a single nucleic acid ligand is identified. Thetarget-specific nucleic acid ligand solution may include a family ofnucleic acid structures or motifs that have a number of conservedsequences and a number of sequences which can be substituted or addedwithout significantly affecting the affinity of the nucleic acid ligandsto the target. By terminating the SELEX process prior to completion, itis possible to determine the sequence of a number of members of thenucleic acid ligand solution family. The invention provides for theidentification of aptamer pools and uses thereof that jointly can beused to characterize a test sample. For example, the aptamer pools canbe identified through rounds of positive and negative selection toidentify cells, tissue or microvesicles indicative of a disease orcondition. The invention further provides use of such aptamer pools tostain, detect and/or quantify such cells, tissue or microvesicles in asample, thereby allowing a diagnosis, prognosis or theranosis to beprovided.

A variety of nucleic acid primary, secondary and tertiary structures areknown to exist. The structures or motifs that have been shown mostcommonly to be involved in non-Watson-Crick type interactions arereferred to as hairpin loops, symmetric and asymmetric bulges,pseudoknots and myriad combinations of the same. Such motifs cantypically be formed in a nucleic acid sequence of no more than 30nucleotides. For this reason, it is often preferred that SELEXprocedures with contiguous randomized segments be initiated with nucleicacid sequences containing a randomized segment of between about 20 toabout 50 nucleotides and in some embodiments, about 30 to about 40nucleotides. In one example, the 5′-fixed:random:3′-fixed sequencecomprises a random sequence of about 30 to about 50 nucleotides. Therandom region may be referred to as the variable region herein.

The core SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. Pat. No. 5,707,796 describes the use ofSELEX in conjunction with gel electrophoresis to select nucleic acidmolecules with specific structural characteristics, such as bent DNA.U.S. Pat. No. 5,763,177 describes SELEX based methods for selectingnucleic acid ligands containing photoreactive groups capable of bindingand/or photocrosslinking to and/or photoinactivating a target molecule.U.S. Pat. Nos. 5,567,588 and 5,861,254 describe SELEX based methodswhich achieve highly efficient partitioning between oligonucleotideshaving high and low affinity for a target molecule. U.S. Pat. No.5,496,938 describes methods for obtaining improved nucleic acid ligandsafter the SELEX process has been performed. U.S. Pat. No. 5,705,337describes methods for covalently linking a ligand to its target.

SELEX can also be used to obtain nucleic acid ligands that bind to morethan one site on the target molecule, and to obtain nucleic acid ligandsthat include non-nucleic acid species that bind to specific sites on thetarget. SELEX provides means for isolating and identifying nucleic acidligands which bind to any envisionable target, including large and smallbiomolecules such as nucleic acid-binding proteins and proteins notknown to bind nucleic acids as part of their biological function as wellas lipids, cofactors and other small molecules. For example, U.S. Pat.No. 5,580,737 discloses nucleic acid sequences identified through SELEXwhich are capable of binding with high affinity to caffeine and theclosely related analog, theophylline.

Counter-SELEX is a method for improving the specificity of nucleic acidligands to a target molecule by eliminating nucleic acid ligandsequences with cross-reactivity to one or more non-target molecules.Counter-SELEX is comprised of the steps of: (a) preparing a candidatemixture of nucleic acids; (b) contacting the candidate mixture with thetarget, wherein nucleic acids having an increased affinity to the targetrelative to the candidate mixture may be partitioned from the remainderof the candidate mixture; (c) partitioning the increased affinitynucleic acids from the remainder of the candidate mixture; (d)dissociating the increased affinity nucleic acids from the target; e)contacting the increased affinity nucleic acids with one or morenon-target molecules such that nucleic acid ligands with specificaffinity for the non-target molecule(s) are removed; and (f) amplifyingthe nucleic acids with specific affinity only to the target molecule toyield a mixture of nucleic acids enriched for nucleic acid sequenceswith a relatively higher affinity and specificity for binding to thetarget molecule. As described above for SELEX, cycles of selection andamplification are repeated until a desired goal is achieved.

A potential problem encountered in the use of nucleic acids astherapeutics and vaccines is that oligonucleotides in theirphosphodiester form may be quickly degraded in body fluids byintracellular and extracellular enzymes such as endonucleases andexonucleases before the desired effect is manifest. The SELEX methodthus encompasses the identification of high-affinity nucleic acidligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX identified nucleic acid ligands containingmodified nucleotides are described, e.g., in U.S. Pat. No. 5,660,985,which describes oligonucleotides containing nucleotide derivativeschemically modified at the 2′ position of ribose, 5′ position ofpyrimidines, and 8′ position of purines, U.S. Pat. No. 5,756,703 whichdescribes oligonucleotides containing various 2′-modified pyrimidines,and U.S. Pat. No. 5,580,737 which describes highly specific nucleic acidligands containing one or more nucleotides modified with 2′-amino(2′-NH₂), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe) substituents.

Modifications of the nucleic acid ligands contemplated in this inventioninclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability,hydrophobicity, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Modifications to generate oligonucleotide populationswhich are resistant to nucleases can also include one or more substituteinternucleotide linkages, altered sugars, altered bases, or combinationsthereof. Such modifications include, but are not limited to, 2′-positionsugar modifications, 5-position pyrimidine modifications, 8-positionpurine modifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbonemodifications, phosphorothioate or allyl phosphate modifications,methylations, and unusual base-pairing combinations such as the isobasesisocytidine and isoguanosine. Modifications can also include 3′ and 5′modifications such as capping.

In one embodiment, oligonucleotides are provided in which the P(O)Ogroup is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), P(O)NR₂(“amidate”), P(O)R, P(O)OR′, CO or CH₂ (“formacetal”) or 3′-amine(—NH—CH₂—CH₂—), wherein each R or R′ is independently H or substitutedor unsubstituted alkyl. Linkage groups can be attached to adjacentnucleotides through an —O—, —N—, or —S— linkage. Not all linkages in theoligonucleotide are required to be identical. As used herein, the termphosphorothioate encompasses one or more non-bridging oxygen atoms in aphosphodiester bond replaced by one or more sulfur atoms.

In further embodiments, the oligonucleotides comprise modified sugargroups, for example, one or more of the hydroxyl groups is replaced withhalogen, aliphatic groups, or functionalized as ethers or amines. In oneembodiment, the 2′-position of the furanose residue is substituted byany of an O-methyl, O-alkyl, O-allyl, S-alkyl, S-allyl, or halo group.Methods of synthesis of 2′-modified sugars are described, e.g., inSproat, et al., Nucl. Acid Res. 19:733-738 (1991); Cotten, et al., Nucl.Acid Res. 19:2629-2635 (1991); and Hobbs, et al., Biochemistry12:5138-5145 (1973). Other modifications are known to one of ordinaryskill in the art. Such modifications may be pre-SELEX processmodifications or post-SELEX process modifications (modification ofpreviously identified unmodified ligands) or may be made byincorporation into the SELEX process.

Pre-SELEX process modifications or those made by incorporation into theSELEX process yield nucleic acid ligands with both specificity for theirSELEX target and improved stability, e.g., in vivo stability. Post-SELEXprocess modifications made to nucleic acid ligands may result inimproved stability, e.g., in vivo stability without adversely affectingthe binding capacity of the nucleic acid ligand.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. Pat. Nos. 5,637,459 and 5,683,867. The SELEX methodfurther encompasses combining selected nucleic acid ligands withlipophilic or non-immunogenic high molecular weight compounds in adiagnostic or therapeutic complex, as described, e.g., in U.S. Pat. Nos.6,011,020, 6,051,698, and PCT Publication No. WO 98/18480. These patentsand applications teach the combination of a broad array of shapes andother properties, with the efficient amplification and replicationproperties of oligonucleotides, and with the desirable properties ofother molecules.

The identification of nucleic acid ligands to small, flexible peptidesvia the SELEX method has also been explored. U.S. Pat. No. 5,648,214identified high affinity RNA nucleic acid ligands to an 11 amino acid.

Aptamers/oligonucleotide probes with desired specificity and bindingaffinity to the target(s) of interest to the present invention can beselected by the SELEX N process as described herein. As part of theSELEX process, the sequences selected to bind to the target are thenoptionally minimized to determine the minimal sequence having thedesired binding affinity. The selected sequences and/or the minimizedsequences are optionally optimized by performing random or directedmutagenesis of the sequence to increase binding affinity oralternatively to determine which positions in the sequence are essentialfor binding activity. Additionally, selections can be performed withsequences incorporating modified nucleotides to stabilize the aptamermolecules against degradation in vivo.

For an aptamer to be suitable for use as a therapeutic, it is preferablyinexpensive to synthesize, and safe and stable in vivo. Wild-type RNAand DNA aptamers are typically not stable is vivo because of theirsusceptibility to degradation by nucleases. Resistance to nucleasedegradation can be greatly increased by the incorporation of modifyinggroups at the 2′-position.

Fluoro and amino groups have been successfully incorporated intooligonucleotide pools from which aptamers have been subsequentlyselected. However, these modifications greatly increase the cost ofsynthesis of the resultant aptamer, and may introduce safety concerns insome cases because of the possibility that the modified nucleotidescould be recycled into host DNA by degradation of the modifiedoligonucleotides and subsequent use of the nucleotides as substrates forDNA synthesis.

Aptamers that contain 2′-O-methyl (“2′-OMe”) nucleotides, as providedherein, may overcome one or more potential drawbacks. Oligonucleotidescontaining 2′-OMe nucleotides are nuclease-resistant and inexpensive tosynthesize. Although 2′-OMe nucleotides are ubiquitous in biologicalsystems, natural polymerases do not accept 2′-OMe NTPs as substratesunder physiological conditions, thus there are no safety concerns overthe recycling of 2′-OMe nucleotides into host DNA. The SELEX method usedto generate 2′-modified aptamers is described, e.g., in U.S. ProvisionalPatent Application Ser. No. 60/430,761, filed Dec. 3, 2002, U.S.Provisional Patent Application Ser. No. 60/487,474, filed Jul. 15, 2003,U.S. Provisional Patent Application Ser. No. 60/517,039, filed Nov. 4,2003, U.S. patent application Ser. No. 10/729,581, filed Dec. 3, 2003,and U.S. patent application Ser. No. 10/873,856, filed Jun. 21, 2004,entitled “Method for in vitro Selection of 2′-O-methyl substitutedNucleic Acids,” each of which is herein incorporated by reference in itsentirety.

Therapeutics

As used herein “therapeutically effective amount” refers to an amount ofa composition that relieves (to some extent, as judged by a skilledmedical practitioner) one or more symptoms of a medical condition suchas a disease or disorder in a subject. Additionally, by “therapeuticallyeffective amount” of a composition is meant an amount that returns tonormal, either partially or completely, physiological or biochemicalparameters associated with or causative of a disease or condition. Aclinician skilled in the art can determine the therapeutically effectiveamount of a composition in order to treat or prevent a particulardisease condition, or disorder when it is administered, such asintravenously, subcutaneously, intraperitoneally, orally, or throughinhalation. The precise amount of the composition required to betherapeutically effective will depend upon numerous factors, e.g., suchas the specific activity of the active agent, the delivery deviceemployed, physical characteristics of the agent, purpose for theadministration, in addition to many patient specific considerations. Buta determination of a therapeutically effective amount is within theskill of an ordinarily skilled clinician upon the appreciation of thedisclosure set forth herein.

The terms “treating,” “treatment,” “therapy,” and “therapeutictreatment” as used herein refer to curative therapy, prophylactictherapy, or preventative therapy. An example of “preventative therapy”is the prevention or lessening the chance of a targeted disease (e.g.,cancer or other proliferative disease) or related condition thereto.Those in need of treatment include those already with the disease orcondition as well as those prone to have the disease or condition to beprevented. The terms “treating,” “treatment,” “therapy,” and“therapeutic treatment” as used herein also describe the management andcare of a mammal for the purpose of combating a disease, or relatedcondition, and includes the administration of a composition to alleviatethe symptoms, side effects, or other complications of the disease,condition. Therapeutic treatment for cancer includes, but is not limitedto, surgery, chemotherapy, radiation therapy, gene therapy, andimmunotherapy.

As used herein, the term “agent” or “drug” or “therapeutic agent” refersto a chemical compound, a mixture of chemical compounds, a biologicalmacromolecule, or an extract made from biological materials such asbacteria, plants, fungi, or animal (particularly mammalian) cells ortissues that are suspected of having therapeutic properties. The agentor drug can be purified, substantially purified or partially purified.An “agent” according to the present invention, also includes a radiationtherapy agent or a “chemotherapuetic agent.”

As used herein, the term “diagnostic agent” refers to any chemical usedin the imaging of diseased tissue, such as, e.g., a tumor.

As used herein, the term “chemotherapuetic agent” refers to an agentwith activity against cancer, neoplastic, and/or proliferative diseases,or that has ability to kill cancerous cells directly.

As used herein, “pharmaceutical formulations” include formulations forhuman and veterinary use with no significant adverse toxicologicaleffect. “Pharmaceutically acceptable formulation” as used herein refersto a composition or formulation that allows for the effectivedistribution of the nucleic acid molecules of the instant invention inthe physical location most suitable for their desired activity.

As used herein the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thecompositions is contemplated.

Aptamer-Toxin Conjugates as a Cancer Therapeutic

Previous work has developed the concept of antibody-toxin conjugates(“immunoconjugates”) as potential therapies for a range of indications,mostly directed at the treatment of cancer with a primary focus onhematological tumors. A variety of different payloads for targeteddelivery have been tested in pre-clinical and clinical studies,including protein toxins, high potency small molecule cytotoxics,radioisotopes, and liposome-encapsulated drugs. While these efforts havesuccessfully yielded several FDA-approved therapies for hematologicaltumors, immunoconjugates as a class (especially for solid tumors) facechallenges that have been attributable to multiple different propertiesof antibodies, including tendencies to develop neutralizing antibodyresponses to non-humanized antibodies, limited penetration in solidtumors, loss of target binding affinity as a result of toxinconjugation, and imbalances between antibody half-life and toxinconjugate half-life that limit the overall therapeutic index (reviewedby Reff and Heard, Critical Reviews in Oncology/Hematology, 40(2001):25-35).

Aptamers are functionally similar to antibodies in target recognition,although their absorption, distribution, metabolism, and excretion(“ADME”) properties are intrinsically different and they generally lackmany of the immune effector functions generally associated withantibodies (e.g., antibody-dependent cellular cytotoxicity,complement-dependent cytotoxicity). In comparing many of the propertiesof aptamers and antibodies previously described, several factors suggestthat toxin-delivery via aptamers offers several concrete advantages overdelivery with antibodies, ultimately affording them better potential astherapeutics. Several examples of the advantages of toxin-delivery viaaptamers over antibodies are as follows:

1) Aptamer-toxin conjugates are entirely chemically synthesized.Chemical synthesis provides more control over the nature of theconjugate. For example, the stoichiometry (ratio of toxins per aptamer)and site of attachment can be precisely defined. Different linkerchemistries can be readily tested. The reversibility of aptamer foldingmeans that loss of activity during conjugation is unlikely and providesmore flexibility in adjusting conjugation conditions to maximize yields.

2) Smaller size allows better tumor penetration. Poor penetration ofantibodies into solid tumors is often cited as a factor limiting theefficacy of conjugate approaches. See Colcher, D., Goel, A., Pavlinkova,G., Beresford, G., Booth, B., Batra, S. K. (1999) “Effects of geneticengineering on the pharmacokinetics of antibodies,” Q. J. Nucl. Med.,43: 132-139. Studies comparing the properties of unPEGylatedanti-tenascin C aptamers with corresponding antibodies demonstrateefficient uptake into tumors (as defined by the tumor:blood ratio) andevidence that aptamer localized to the tumor is unexpectedly long-lived(t_(1/2)>12 hours) (Hicke, B. J., Stephens, A. W., “Escort aptamers: adelivery service for diagnosis and therapy”, J. Clin. Invest.,106:923-928 (2000)).

3) Tunable PK. Aptamer half-life/metabolism can be more easily tuned tomatch properties of payload, optimizing the ability to deliver toxin tothe tumor while minimizing systemic exposure. Appropriate modificationsto the aptamer backbone and addition of high molecular weight PEGsshould make it possible to match the half-life of the aptamer to theintrinsic half-life of the conjugated toxin/linker, minimizing systemicexposure to non-functional toxin-bearing metabolites (expected ift_(1/2)(aptamer)<<t_(1/2)(toxin)) and reducing the likelihood thatpersisting unconjugated aptamer will functionally block uptake ofconjugated aptamer (expected if t_(1/2)(aptamer)>>t_(1/2) (toxin)).

4) Relatively low material requirements. It is likely that dosing levelswill be limited by toxicity intrinsic to the cytotoxic payload. As such,a single course of treatment will likely entail relatively small (<100mg) quantities of aptamer, reducing the likelihood that the cost ofoligonucleotide synthesis will be a barrier for aptamer-based therapies.

5) Parenteral administration is preferred for this indication. Therewill be no special need to develop alternative formulations to drivepatient/physician acceptance.

The invention provides a pharmaceutical composition comprising atherapeutically effective amount of an aptamer provided by the inventionor a salt thereof, and a pharmaceutically acceptable carrier or diluent.The invention also provides a pharmaceutical composition comprising atherapeutically effective amount of the aptamer or a salt thereof, and apharmaceutically acceptable carrier or diluent. Relatedly, the inventionprovides a method of treating or ameliorating a disease or disorder,comprising administering the pharmaceutical composition to a subject inneed thereof. Administering a therapeutically effective amount of thecomposition to the subject may result in: (a) an enhancement of thedelivery of the active agent to a disease site relative to delivery ofthe active agent alone; or (b) an enhancement of microvesicles clearanceresulting in a decrease of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% in a blood level of microvesicles targeted by the aptamer;or (c) an decrease in biological activity of microvesicles targeted bythe aptamer of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.In an embodiment, the biological activity of microvesicles comprisesimmune suppression or transfer of genetic information. The disease ordisorder can include without limitation those disclosed herein. Forexample, the disease or disorder may comprise a neoplastic,proliferative, or inflammatory, metabolic, cardiovascular, orneurological disease or disorder. See, e.g., section “Phenotypes.”

Anti-Target and Multivalent Oligonucleotides

As described herein, the target of oligonucleotide probes can beidentified. For example, when the target comprises a protein or proteincomplex (e.g., a nucleoprotein or lipoprotein), identifying the targetmay comprise use of mass spectrometry (MS), peptide mass fingerprinting(PMF; protein fingerprinting), sequencing, N-terminal amino acidanalysis, C-terminal amino acid analysis, Edman degradation,chromatography, electrophoresis, two-dimensional gel electrophoresis (2Dgel), antibody array, or immunoassay. Such approaches can be applied toidentify a number of targets recognized by an oligonucleotide probelibrary. For example, an oligonucleotide probe library can be incubatedwith a sample of interest, bound members of the library captured, andthe targets bound to the captured members identified. See Example 9herein for an example of such target identification using massspectrometry.

The oligonucleotide aptamers to the various targets can be used formultiple purposes. In some embodiments, the aptamers are used astherapeutic agents Immunotherapeutic approaches using antibodies thatrecognize foreign/misfolded antigens (e.g., anti-CD20, anti-CD30,anti-CD33, anti-CD52, anti-EGFR, anti-nucleolin, anti-nucleophosmin,etc.) can selectively kill target cells via linked therapeutic agents orby stimulating the immune system through activation of cell-mediatedcytotoxicity. Aptamers or oligonucleotides are an attractiveimmunotherapeutic alternative for various reasons such as low cost,small size, ease and speed of synthesis, stability and lowimmunogenicity. In an embodiment, immunotherapeutic agents areconjugated to disease specific target oligonucleotide or antibody (Ab)for targeted cell killing via recruitment of complement proteins and thedownstream membrane attack complex. See, e.g., Zhou and Rossi,Cell-type-specific, Aptamer-functionalized Agents for Targeted DiseaseTherapy, Mol Ther Nucleic Acids. 2014 Jun. 17; 3:e169. doi:10.1038/mtna.2014.21; Pei et al., Clinical applications of nucleic acidaptamers in cancer, Mol Clin Oncol. 2014 May; 2(3):341-348. Epub 2014Feb. 10. This approach can be applied to target diseased host cells suchas cancer cells, gram negative bacteria, viral and/or parasiticinfections, and the like.

In some embodiments, the invention provides a multipartite constructcomprising a binding agent specific to a biological target with anotherbinding agent specific to immunomodulatory entity. Examples of suchconstructs are shown in FIG. 8. In Design 1 in the figure, thehorizontal line indicates an oligonucleotide construct, which constructcomprises a 5′ primer 801 (Primer 1), a variable region 802 that can bean aptamer to a target of interest, a 3′ primer 803 (Primer 2), and animmunomodulatory domain region (“IMD”) 804. The complete Design 1construct can be used to bring a target of interest in proximity with animmunomodulatory agent. The primers can be designed for any desiredpurpose, e.g., amplification, capture, modification, direct or indirectlabeling, and the like. In some embodiments, the target of the variableregion is a disease marker and thus the construct is targeted to adiseased tissue, cell or microvesicle. The immunomodulatory domainregion can act as an immune stimulator or suppressor. Any appropriateimmune stimulator or suppressor can be used, e.g., a small molecule,antibody or an aptamer. Thus, the construct can modulate the immuneresponse at a target of interest, e.g., at a cell or microvesiclecarrying the target. The basic construct can be modified as desired. Forexample, Design 2 in FIG. 8 shows the construct carrying a linker 805between Primer 2 803 and the IMD 804. Such linkers are explained furtherbelow and can be inserted between any components of the construct asdesired. Linkers can provide a desired space between the regions of theconstruct and can be manipulated to influence other properties such asstability. Design 3 in FIG. 8 shows another example wherein the IMD 804is an oligonucleotide and the variable region 802 and IMD 804 liebetween the primers 801 and 803. One of skill will appreciate that oneor more linker, such as 805 of Design 2, can also be inserted intoDesign 3, e.g., between the variable region 802 and IMD 804. One ofskill will further appreciate that the ordering of the oligonucleotidesegments from 5′ to 3′ can be modified, e.g., reversed.

As noted, the multipartite constructs may be synthesized and/or modifiedas desired. In some embodiments of the invention, the multipartiteoligonucleotide construct is synthesized directly with or without alinker in between the oligonucleotide segments. See, e.g., FIG. 8 Design3, which can be generated directly via amplification by Primer 1 801 andPrimer 2 803. One or more linker can act as a spacer to create a desiredspacing between the target of the variable region segment 802 and thetarget of the IMD segment 804. The spacing can be determined viacomputer modeling or via experimentation due to steric hindrance orother considerations.

The multipartite constructs can be generated against any appropriatetarget. The targets can include without limitation tumors or diseasedtissues, cells, cancer cells, circulating tumor cells (CTCs), immunecells (e.g., B-cells, T-cells, macrophages, dendritic cells),microvesicles, bacteria, viruses or other parasites. The target can belarge biological complexes, e.g., protein complexes, ribonucleoproteincomplexes, lipid complexes, or a combination thereof. It will beunderstood that the specific target of the multipartite constructs canbe a certain member of the foregoing macromolecular targets. Forexample, consider that the desired target of the multipartite constructis a cell or microvesicle. In such case, the multipartite construct canbe directed to a specific biomarker, e.g., a surface antigen, of thecell or microvesicle. As a non-limiting example, the target of interestcan be B-cells and the specific target of the variable region of themultipartite construct can be CD20. CD20 is a cellular marker of B-cellstargeted by the monoclonal antibodies (mAb) rituximab, obinutuzumab,ofatumumab, ibritumomab tiuxetan, and tositumomab, which are used asagents in the treatment of B-cell lymphomas and leukemias. As anothernon-limiting example, the target of interest can be cancer cells and thespecific target of the variable region of the multipartite construct canbe c-MET. MET is a membrane receptor that is essential for embryonicdevelopment and wound healing. Abnormal MET activation in cancercorrelates with poor prognosis, where aberrantly active MET triggerstumor growth, formation of new blood vessels (angiogenesis), and cancerspread to other organs (metastasis). MET has been observed to bederegulated in many types of human malignancies, including cancers ofkidney, liver, stomach, breast, and brain. Other biomarkers can be usedas the specific target as desired. For example, the biomarker can beselected from any of Tables 3-4, or 10-17 herein, or Table 4 ofInternational Patent Application PCT/US2016/040157, filed Jun. 29, 2016.

As noted above, the IDM domain can be constructed to illicit acomplement mediated immune response that can induce apoptosis. Such IDMcan include but are not limited to C1q, C1r, C1s, C1, C3a, C3b, C3d,C5a, C2, C4, and cytokines. The IDM region may comprise anoligonucleotide sequence including without limitation Toll-Like Receptor(TLR) agonists like CpG sequences which are immunostimulatory and/orpolyG sequences which can be anti-proliferative or pro-apoptotic. Themoiety can be vaccine like moiety or antigen that stimulates an immuneresponse. In an embodiment, the immune stimulating moiety comprises asuperantigen. In some embodiments, the superantigen can be selected fromthe group consisting of staphylococcal enterotoxins (SEs), aStreptococcus pyogenes exotoxin (SPE), a Staphylococcus aureus toxicshock-syndrome toxin (TSST-1), a streptococcal mitogenic exotoxin (SME),a streptococcal superantigen (SSA), a hepatitis surface antigen, or acombination thereof. Other bacterial antigens that can be used with theinvention comprise bacterial antigens such as Freund's completeadjuvant, Freund's incomplete adjuvant, monophosphoryl-lipid A/trehalosedicorynomycolate (Ribi's adjuvant), BCG (Calmette-Guerin Bacillus;Mycobacterium bovis), and Corynebacterium parvum. The immune stimulatingmoiety can also be a non-specific immunostimulant, such as an adjuvantor other non-specific immunostimulator. Useful adjuvants comprisewithout limitation aluminium salts, alum, aluminium phosphate, aluminiumhydroxide, squalene, oils, MF59, and AS03 (“Adjuvant System 03”). Theadjuvant can be selected from the group consisting of Cationicliposome-DNA complex JVRS-100, aluminum hydroxide vaccine adjuvant,aluminum phosphate vaccine adjuvant, aluminum potassium sulfateadjuvant, Alhydrogel, ISCOM(s)™, Freund's Complete Adjuvant, Freund'sIncomplete Adjuvant, CpG DNA Vaccine Adjuvant, Cholera toxin, Choleratoxin B subunit, Liposomes, Saponin Vaccine Adjuvant, DDA Adjuvant,Squalene-based Adjuvants, Etx B subunit Adjuvant, IL-12 VaccineAdjuvant, LTK63 Vaccine Mutant Adjuvant, TiterMax Gold Adjuvant, RibiVaccine Adjuvant, Montanide ISA 720 Adjuvant, Corynebacterium-derivedP40 Vaccine Adjuvant, MPL™ Adjuvant, AS04, AS02, LipopolysaccharideVaccine Adjuvant, Muramyl Dipeptide Adjuvant, CRL1005, KilledCorynebacterium parvum Vaccine Adjuvant, Montanide ISA 51, Bordetellapertussis component Vaccine Adjuvant, Cationic Liposomal VaccineAdjuvant, Adamantylamide Dipeptide Vaccine Adjuvant, Arlacel A, VSA-3Adjuvant, Aluminum vaccine adjuvant, Polygen Vaccine Adjuvant, Adjumer™,Algal Glucan, Bay R1005, Theramide®, Stearyl Tyrosine, Specol,Algammulin, Avridine®, Calcium Phosphate Gel, CTA1-DD gene fusionprotein, DOC/Alum Complex, Gamma Inulin, Gerbu Adjuvant, GM-CSF, GMDP,Recombinant hIFN-gamma/Interferon-g, Interleukin-1β, Interleukin-2,Interleukin-7, Sclavo peptide, Rehydragel LV, Rehydragel HPA,Loxoribine, MF59, MTP-PE Liposomes, Murametide, Murapalmitine,D-Murapalmitine, NAGO, Non-Ionic Surfactant Vesicles, PMMA, ProteinCochleates, QS-21, SPT (Antigen Formulation), nanoemulsion vaccineadjuvant, AS03, Quil-A vaccine adjuvant, RC529 vaccine adjuvant, LTR192GVaccine Adjuvant, E. coli heat-labile toxin, LT, amorphous aluminumhydroxyphosphate sulfate adjuvant, Calcium phosphate vaccine adjuvant,Montanide Incomplete Seppic Adjuvant, Imiquimod, Resiquimod, AF03,Flagellin, Poly(I:C), ISCOMATRIX®, Abisco-100 vaccine adjuvant,Albumin-heparin microparticles vaccine adjuvant, AS-2 vaccine adjuvant,B7-2 vaccine adjuvant, DHEA vaccine adjuvant, Immunoliposomes ContainingAntibodies to Costimulatory Molecules, SAF-1, Sendai Proteoliposomes,Sendai-containing Lipid Matrices, Threonyl muramyl dipeptide (TMDP), TyParticles vaccine adjuvant, Bupivacaine vaccine adjuvant, DL-PGL(Polyester poly (DL-lactide-co-glycolide)) vaccine adjuvant, IL-15vaccine adjuvant, LTK72 vaccine adjuvant, MPL-SE vaccine adjuvant,non-toxic mutant E112K of Cholera Toxin mCT-E112K, and Matrix-S.Additional adjuvants that can be used with the multipartite constructsof the invention can be identified using the Vaxjo database. See SayersS, Ulysse G, Xiang Z, and He Y. Vaxjo: a web-based vaccine adjuvantdatabase and its application for analysis of vaccine adjuvants and theiruses in vaccine development. Journal of Biomedicine and Biotechnology.2012; 2012:831486. Epub 2012 Mar. 13. PMID: 22505817;www.violinet.org/vaxjo/. Other useful non-specific immunostimulatorscomprise histamine, interferon, transfer factor, tuftsin, interleukin-1,female sex hormones, prolactin, growth hormone vitamin D, deoxycholicacid (DCA), tetrachlorodecaoxide (TCDO), and imiquimod or resiquimod,which are drugs that activate immune cells through the toll-likereceptor 7. A multipartite construct can be created that comprises morethan one immunomodulating moiety, e.g., using segments that span CpGsequences which are immunostimulatory with complement directed segmentsthat can stimulate apoptosis.

Modifications

Modifications to the one or more oligonucleotide of the invention can bemade to alter desired characteristics, including without limitation invivo stability, specificity, affinity, avidity or nucleasesusceptibility. Alterations to the half life may improve stability invivo or may reduce stability to limit in vivo toxicity. Such alterationscan include mutations, truncations or extensions. The 5′ and/or 3′ endsof the multipartite oligonucleotide constructs can be protected ordeprotected to modulate stability as well. Modifications to improve invivo stability, specificity, affinity, avidity or nucleasesusceptibility or alter the half life to influence in vivo toxicity maybe at the 5′ or 3′ end and include but are not limited to the following:locked nucleic acid (LNA) incorporation, unlocked nucleic acid (UNA)incorporation, phosphorothioate backbone instead of phosphodiesterbackbone, amino modifiers (i.e. C6-dT), dye conjugates (Cy dues,Fluorophores, etc), Biotinylation, PEG linkers, Click chemistry linkers,dideoxynucleotide end blockers, inverted end bases, cholesterol TEG orother lipid based labels.

Linkage options for segments of the oligonucleotide of the invention canbe on the 5′ or 3′ end of an oligonucleotide or to a primary amine,sulfhydryl or carboxyl group of an antibody and include but are notlimited to the following: Biotin-target oligonucleotide/Ab,streptavidin-complement oligonucleotide or vice versa, aminomodified-target Ab/oligonucleotide, thiol/carboxy-complementoligonucleotide or vice versa, Click chemistry-targetAb/oligonucleotide, corresponding Click chemistry partner-complementoligonucleotide or vice versa. The linkages may be covalent ornon-covalent and may include but are not limited to monovalent,multivalent (i.e. bi, tri or tetra-valent) assembly, to a DNA scaffold(i.e. DNA origami structure), drug/chemotherapeutic agent, nanoparticle,microparticle or a micelle or liposome.

A linker region can comprise a spacer with homo- or multifunctionalreactive groups that can vary in length and type. These include but arenot limited to the following: spacer C18, PEG4, PEG6, PEG8, and PEG12.

The multipartite oligonucleotide of the invention can further compriseadditional elements to add desired biological effects. For example, theoligonucleotide of the invention may comprise a membrane disruptivemoiety. The oligonucleotide of the invention may also be conjugated toone or more chemical moiety that provides such effects. For example, theoligonucleotide of the invention may be conjugated to a detergent-likemoiety to disrupt the membrane of a target cell or microvesicle. Usefulionic detergents include sodium dodecyl sulfate (SDS, sodium laurylsulfate (SLS)), sodium laureth sulfate (SLS, sodium lauryl ether sulfate(SLES)), ammonium lauryl sulfate (ALS), cetrimonium bromide, cetrimoniumchloride, cetrimonium stearate, and the like. Useful non-ionic(zwitterionic) detergents include polyoxyethylene glycols, polysorbate20 (also known as Tween 20), other polysorbates (e.g., 40, 60, 65, 80,etc), Triton-X (e.g., X100, X114),3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),CHAPSO, deoxycholic acid, sodium deoxycholate, NP-40, glycosides,octyl-thio-glucosides, maltosides, and the like. One of skill willappreciate that functional fragments, such as membrance disruptivemoieties, can be covalently or non-covalently attached to theoligonucleotide of the invention.

Oligonucleotide segments, including those of a multipartite construct,can include any desirable base modification known in the art. In certainembodiments, oligonucleotide segments are 10 to 50 nucleotides inlength. One having ordinary skill in the art will appreciate that thisembodies oligonucleotides of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length,or any range derivable there within.

In certain embodiments, a multipartite construct comprises a chimericoligonucleotide that contains two or more chemically distinct regions,each made up of at least one nucleotide. Such chimeras can be referredto using terms such as multipartite, multivalent, or the like. Theoligonucleotides portions may contain at least one region of modifiednucleotides that confers one or more beneficial properties, e.g.,increased nuclease resistance, bioavailability, increased bindingaffinity for the target. Chimeric nucleic acids of the invention may beformed as composite structures of two or more oligonucleotides, two ormore types of oligonucleotides (e.g., both DNA and RNA segments),modified oligonucleotides, oligonucleosides and/or oligonucleotidemimetics. Such compounds have also been referred to in the art ashybrids. Representative United States patents that teach the preparationof such hybrid structures comprise, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference in its entirety.

In certain embodiments, an oligonucleotide of the invention comprises atleast one nucleotide modified at the 2′ position of the sugar, includingwithout limitation a 2′-0-alkyl, 2′-0-alkyl-0-alkyl or2′-fluoro-modified nucleotide. In other embodiments, RNA modificationsinclude 2′-fluoro, 2′-amino and 2′ O-methyl modifications on the riboseof pyrimidines, a basic residue or an inverted base at the 3′ end of theRNA. Such modifications are routinely incorporated into oligonucleotidesand these oligonucleotides have been shown to have higher target bindingaffinity in some cases than 2′-deoxyoligonucleotides against a giventarget.

A number of nucleotide and nucleoside modifications have been shown tomake an oligonucleotide more resistant to nuclease digestion, therebyprolonging in vivo half-life. Specific examples of modifiedoligonucleotides include those comprising backbones comprising, forexample, phosphorothioates, phosphotriesters, methyl phosphonates, shortchain alkyl or cycloalkyl intersugar linkages or short chainheteroatomic or heterocyclic intersugar linkages. The constructs of theinvention can comprise oligonucleotides with phosphorothioate backbonesand/or heteroatom backbones, e.g., CH2-NH-0-CH2, CH, ˜N(CH3)˜0˜CH2(known as a methylene(methylimino) or MMI backbone], CH2-O—N (CH3)-CH2,CH2-N (CH3)-N (CH3)-CH2 and O—N (CH3)-CH2-CH2 backbones, wherein thenative phosphodiester backbone is represented as O—P—O—CH); amidebackbones (De Mesmaeker et ah, 1995); morpholino backbone structures(Summerton and Weller, U.S. Pat. No. 5,034,506); peptide nucleic acid(PNA) backbone (wherein the phosphodiester backbone of theoligonucleotide is replaced with a polyamide backbone, the nucleotidesbeing bound directly or indirectly to the aza nitrogen atoms of thepolyamide backbone (Nielsen, et al., 1991), each of which is hereinincorporated by reference in its entirety. Phosphorus-containinglinkages include, but are not limited to, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatescomprising 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates comprising 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3*-5* to 5*-3* or 2*-5* to 5*-2*; see U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5, 177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321, 131;5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference in its entirety. Morpholino-based oligomeric compounds areknown in the art described in Braasch & Corey, Biochemistry vol. 41, no.14, 2002, pages 4503-4510; Genesis vol. 30, 2001, page 3; Heasman, J.Dev. Biol. vol. 243, 2002, pages 209-214; Nasevicius et al. Nat. Genet.vol. 26, 2000, pages 216-220; Lacerra et al. Proc. Natl. Acad. Sci. vol.97, 2000, pages 9591-9596 and U.S. Pat. No. 5,034,506, issued Jul. 23,1991, each of which is herein incorporated by reference in its entirety.Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., J. Am. Chem. Soc. Vol. 122, 2000, pages 8595-8602, the contentsof which is incorporated herein in its entirety. An oligonucleotide ofthe invention can comprise at least such modification as desired.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that can be formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216, 141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, each of which is herein incorporated by reference in itsentirety. An oligonucleotide of the invention can comprise at least suchmodification as desired.

In certain embodiments, an oligonucleotide of the invention comprisesone or more substituted sugar moieties, e.g., one of the following atthe 2′ position: OH, SH, SCH₃, F, OCN, OCH₃ OCH₃, OCH₃ O(CH₂)n CH₃,O(CH₂)n NH₂ or O(CH₂)n CH₃ where n is from 1 to about 10; Ci to CIOlower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl;CI; Br; CN; CF₃; OCF₃; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH₃;SO₂ CH₃; ONO₂; N O₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleavinggroup; a reporter group; an intercalator; a group for improving thepharmacokinetic properties of an oligonucleotide; or a group forimproving the pharmacokinetic/pharmacodynamic properties of anoligonucleotide and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy [2′-0-CH2CH2OCH3, alsoknown as 2′-0-(2-methoxyethyl)]. Other preferred modifications include2*-methoxy (2*-0-CH3), 2*-propoxy (2*-OCH2 CH2CH3) and 2*-fluoro (2*-F)Similar modifications may also be made at other positions on theoligonucleotide, e.g., the 3′ position of the sugar on the 3′ terminalnucleotide and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutyls inplace of the pentofuranosyl group.

In certain embodiments, an oligonucleotide of the invention comprisesone or more base modifications and/or substitutions. As used herein,“unmodified” or “natural” bases include adenine (A), guanine (G),thymine (T), cytosine (C) and uracil (U). Modified bases include,without limitation, bases found only infrequently or transiently innatural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Mepyrimidines, particularly 5-methylcytosine (also referred to as5-methyl-2′ deoxy cytosine and often referred to in the art as 5-Me-C),5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as wellas synthetic bases, e.g., 2-aminoadenine, 2-(methylamino)adenine,2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or otherheterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine,5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine and 2,6-diaminopurine (Kornberg, 1980; Gebeyehu,et ah, 1987). A “universal” base known in the art, e.g., inosine, canalso be included. 5-Me-C substitutions can also be included. These havebeen shown to increase nucleic acid duplex stability by 0.6-1.20C. See,e.g., Sanghvi et al., ‘Antisense Research & Applications’, 1993, CRCPRESS pages 276-278. Further suitable modified bases are described inU.S. Pat. Nos. 3,687,808, as well as 4,845,205; 5,130,302; 5,134,066;5,175, 273; 5, 367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617;5,750,692, and 5,681,941, each of which is herein incorporated byreference.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even atwithin a single nucleoside within an oligonucleotide.

In certain embodiments, both a sugar and an internucleoside linkage,i.e., the backbone, of one or more nucleotide units within anoligonucleotide of the invention are replaced with novel groups. Thebase can be maintained for hybridization with an appropriate nucleicacid target compound. One such oligomeric compound, an oligonucleotidemimetic that has been shown to retain hybridization properties, isreferred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar-backbone of an oligonucleotide is replaced with an amidecontaining backbone, for example, an aminoethylglycine backbone. Thenucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. Representativepatents that teach the preparation of PNA compounds comprise, but arenot limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, eachof which is herein incorporated by reference. Further teaching of PNAcompounds can be found in Nielsen et al. Science vol. 254, 1991, page1497, which is herein incorporated by reference.

In certain embodiments, the oligonucleotide of the invention is linked(covalently or non-covalently) to one or more moieties or conjugatesthat enhance activity, cellular distribution, or localization. Suchmoieties include, without limitation, lipid moieties such as acholesterol moiety (Letsinger et al. Proc. Natl. Acad. Sci. Usa. vol.86, 1989, pages 6553-6556), cholic acid (Manoharan et al. Bioorg. Med.Chem. Let. vol. 4, 1994, pages 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al. Ann. N. Y. Acad. Sci. Vol. 660,1992, pages 306-309; Manoharan et al. Bioorg. Med. Chem. Let. vol. 3,1993, pages 2765-2770), a thiocholesterol (Oberhauser et al. Nucl. AcidsRes. vol. 20, 1992, pages 533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Kabanov et al. Febs Lett. vol. 259,1990, pages 327-330; Svinarchuk et al. Biochimie vol. 75, 1993, pages49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al. Tetrahedron Lett. vol. 36, 1995, pages 3651-3654; Sheaet al. Nucl. Acids Res. vol. 18, 1990, pages 3777-3783), a polyamine ora polyethylene glycol chain (Mancharan et al. Nucleosides & Nucleotidesvol. 14, 1995, pages 969-973), or adamantane acetic acid (Manoharan etal. Tetrahedron Lett. vol. 36, 1995, pages 3651-3654), a palmityl moiety(Mishra et al. Biochim. Biophys. Acta vol. 1264, 1995, pages 229-237),or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety(Crooke et al. J. Pharmacol. Exp. Ther. vol. 277, 1996, pages 923-937),each of which is herein incorporated by reference in its entirety. Seealso U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465;5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731;5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603;5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250;5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463;5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142;5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and5,688,941, each of which is herein incorporated by reference in itsentirety.

The oligonucleotide of the invention can be modified to incorporate awide variety of modified nucleotides as desired. For example, theconstruct may be synthesized entirely of modified nucleotides or with asubset of modified nucleotides. The modifications can be the same ordifferent. Some or all nucleotides may be modified, and those that aremodified may contain the same modification. For example, all nucleotidescontaining the same base may have one type of modification, whilenucleotides containing other bases may have different types ofmodification. All purine nucleotides may have one type of modification(or are unmodified), while all pyrimidine nucleotides have another,different type of modification (or are unmodified). Thus, the constructmay comprise any combination of desired modifications, including forexample, ribonucleotides (2′-OH), deoxyribonucleotides (2′-deoxy),2′-amino nucleotides (2′-NH2), 2′-fluoro nucleotides (2′-F) and2′-0-methyl (2′-OMe) nucleotides.

In some embodiments, the oligonucleotide of the invention is synthesizedusing a transcription mixture containing modified nucleotides in orderto generate a modified construct. For example, a transcription mixturemay contain only 2′-OMe A, G, C and U and/or T triphosphates (2′-OMeATP, 2′-OMe UTP and/or 2*-OMe TTP, 2*-OMe CTP and 2*-OMe GTP), referredto as an MNA or mRmY mixture. Oligonucleotides generated therefrom arereferred to as MNA oligonucleotides or mRmY oligonucleotides and containonly 2′-0-methyl nucleotides. A transcription mixture containing all2′-OH nucleotides is referred to as an “rN” mixture, andoligonucleotides generated therefrom are referred to as “rN”, “rRrY” orRNA oligonucleotides. A transcription mixture containing all deoxynucleotides is referred to as a “dN” mixture, and oligonucleotidesgenerated therefrom are referred to as “dN”, “dRdY” or DNAoligonucleotides. Alternatively, a subset of nucleotides (e.g., C, Uand/or T) may comprise a first modified nucleotides (e.g, 2′-OMe)nucleotides and the remainder (e.g., A and G) comprise a second modifiednucleotide (e.g., 2′-OH or 2′-F). For example, a transcription mixturecontaining 2′-F U and 2′-OMe A, G and C is referred to as a “fUmV”mixture, and oligonucleotides generated therefrom are referred to as“fUmV” oligonucleotides. A transcription mixture containing 2′-F A andG, and 2′-OMe C and U and/or T is referred to as an “fRmY” mixture, andoligonucleotides generated therefrom are referred to as “fRmY”oligonucleotides. A transcription mixture containing 2′-F A and 2′-OMeC, G and U and/or T is referred to as “fAmB” mixture, andoligonucleotides generated therefrom are referred to as “fAmB”oligonucleotides.

One of skill in the art can improve pre-identified aptamer segments(e.g., variable regions or immunomodulatory regions that comprise anaptamer to a biomarker target or other entity) using various processmodifications. Examples of such process modifications include, but arenot limited to, truncation, deletion, substitution, or modification of asugar or base or internucleotide linkage, capping, and PEGylation. Inaddition, the sequence requirements of an aptamer may be exploredthrough doped reselections or aptamer medicinal chemistry. Dopedreselections are carried out using a synthetic, degenerate pool that hasbeen designed based on the aptamer of interest. The level of degeneracyusually varies from about 70-85% from the aptamer of interest. Ingeneral, sequences with neutral mutations are identified through thedoped reselection process. Aptamer medicinal chemistry is an aptamerimprovement technique in which sets of variant aptamers are chemicallysynthesized. These variants are then compared to each other and to theparent aptamer. Aptamer medicinal chemistry is used to explore thelocal, rather than global, introduction of substituents. For example,the following modifications may be introduced: modifications at a sugar,base, and/or internucleotide linkage, such as 2′-deoxy, 2′-ribo, or2′-0-methyl purines or pyrimidines, phosphorothioate linkages may beintroduced between nucleotides, a cap may be introduced at the 5′ or 3′end of the aptamer (such as 3′ inverted dT cap) to block degradation byexonucleases, or a polyethylene glycol (PEG) element may be added to theaptamer to increase the half-life of the aptamer in the subject.

Additional compositions comprising an oligonucleotide of the inventionand uses thereof are further described below. As the invention providesmethods to identify oligonucleotide probes that bind to specifictissues, cells, microvesicles or other biological entities of interest,the oligonucleotide probes of the invention target such entities and areinherently drug candidates, agents that can be used for targeted drugdelivery, or both.

Pharmaceutical Compositions

In an aspect, the invention provides pharmaceutical compositionscomprising one or more oligonucleotide of the invention, e.g., as astandalone drug, as a drug delivery agent, as a multipartite constructas described above, or any combination thereof. The invention furtherprovides methods of administering such compositions.

The term “condition,” as used herein means an interruption, cessation,or disorder of a bodily function, system, or organ. Representativeconditions include, but are not limited to, diseases such as cancer,inflammation, diabetes, and organ failure.

The phrase “treating,” “treatment of,” and the like include theamelioration or cessation of a specified condition.

The phrase “preventing,” “prevention of,” and the like include theavoidance of the onset of a condition.

The term “salt,” as used herein, means two compounds that are notcovalently bound but are chemically bound by ionic interactions.

The term “pharmaceutically acceptable,” as used herein, when referringto a component of a pharmaceutical composition means that the component,when administered to an animal, does not have undue adverse effects suchas excessive toxicity, irritation, or allergic response commensuratewith a reasonable benefit/risk ratio. Accordingly, the term“pharmaceutically acceptable organic solvent,” as used herein, means anorganic solvent that when administered to an animal does not have undueadverse effects such as excessive toxicity, irritation, or allergicresponse commensurate with a reasonable benefit/risk ratio. Preferably,the pharmaceutically acceptable organic solvent is a solvent that isgenerally recognized as safe (“GRAS”) by the United States Food and DrugAdministration (“FDA”). Similarly, the term “pharmaceutically acceptableorganic base,” as used herein, means an organic base that whenadministered to an animal does not have undue adverse effects such asexcessive toxicity, irritation, or allergic response commensurate with areasonable benefit/risk ratio.

The phrase “injectable” or “injectable composition,” as used herein,means a composition that can be drawn into a syringe and injectedsubcutaneously, intraperitoneally, or intramuscularly into an animalwithout causing adverse effects due to the presence of solid material inthe composition. Solid materials include, but are not limited to,crystals, gummy masses, and gels. Typically, a formulation orcomposition is considered to be injectable when no more than about 15%,preferably no more than about 10%, more preferably no more than about5%, even more preferably no more than about 2%, and most preferably nomore than about 1% of the formulation is retained on a 0.22 μm filterwhen the formulation is filtered through the filter at 98° F. There are,however, some compositions of the invention, which are gels, that can beeasily dispensed from a syringe but will be retained on a 0.22 μmfilter. In one embodiment, the term “injectable,” as used herein,includes these gel compositions. In one embodiment, the term“injectable,” as used herein, further includes compositions that whenwarmed to a temperature of up to about 40° C. and then filtered througha 0.22 μm filter, no more than about 15%, preferably no more than about10%, more preferably no more than about 5%, even more preferably no morethan about 2%, and most preferably no more than about 1% of theformulation is retained on the filter. In one embodiment, an example ofan injectable pharmaceutical composition is a solution of apharmaceutically active compound (for example, one or moreoligonucleotide of the invention, e.g., a multipartite construct, ananti-C1Q oligonucleotide, a 10.36 oligonucleotide, as described above,or any combination thereof) in a pharmaceutically acceptable solvent.One of skill will appreciate that injectable solutions have inherentproperties, e.g., sterility, pharmaceutically acceptable excipients andfree of harmful measures of pyrogens or similar contaminants.

The term “solution,” as used herein, means a uniformly dispersed mixtureat the molecular or ionic level of one or more substances (solute), inone or more other substances (solvent), typically a liquid.

The term “suspension,” as used herein, means solid particles that areevenly dispersed in a solvent, which can be aqueous or non-aqueous.

The term “animal,” as used herein, includes, but is not limited to,humans, canines, felines, equines, bovines, ovines, porcines,amphibians, reptiles, and avians. Representative animals include, butare not limited to a cow, a horse, a sheep, a pig, an ungulate, achimpanzee, a monkey, a baboon, a chicken, a turkey, a mouse, a rabbit,a rat, a guinea pig, a dog, a cat, and a human. In one embodiment, theanimal is a mammal. In one embodiment, the animal is a human. In oneembodiment, the animal is a non-human. In one embodiment, the animal isa canine, a feline, an equine, a bovine, an ovine, or a porcine.

The phrase “drug depot,” as used herein means a precipitate, whichincludes one or more oligonucleotide of the invention, e.g., amultipartite construct, an anti-C1Q oligonucleotide, a 10.36oligonucleotide, as described above, or any combination thereof, formedwithin the body of a treated animal that releases the oligonucleotideover time to provide a pharmaceutically effective amount of theoligonucleotide.

The phrase “substantially free of,” as used herein, means less thanabout 2 percent by weight. For example, the phrase “a pharmaceuticalcomposition substantially free of water” means that the amount of waterin the pharmaceutical composition is less than about 2 percent by weightof the pharmaceutical composition.

The term “effective amount,” as used herein, means an amount sufficientto treat or prevent a condition in an animal.

The nucleotides that make up the oligonucleotide of the invention can bemodified to, for example, improve their stability, i.e., improve theirin vivo half-life, and/or to reduce their rate of excretion whenadministered to an animal. The term “modified” encompasses nucleotideswith a covalently modified base and/or sugar. For example, modifiednucleotides include nucleotides having sugars which are covalentlyattached to low molecular weight organic groups other than a hydroxylgroup at the 3′ position and other than a phosphate group at the 5′position. Modified nucleotides may also include 2′ substituted sugarssuch as 2′-O-methyl-; 2′-O-alkyl; 2′-O-allyl; 2′-S-alkyl; 2′-S-allyl;2′-fluoro-; 2′-halo or 2′-azido-ribose; carbocyclic sugar analogues;α-anomeric sugars; and epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, and sedoheptulose.

Modified nucleotides are known in the art and include, but are notlimited to, alkylated purines and/or pyrimidines; acylated purinesand/or pyrimidines; or other heterocycles. These classes of pyrimidinesand purines are known in the art and include, pseudoisocytosine;N4,N4-ethanocytosine; 8-hydroxy-N6-methyladenine; 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil; 5-fluorouracil; 5-bromouracil;5-carboxymethylaminomethyl-2-thiouracil; 5-carboxymethylaminomethyluracil; dihydrouracil; inosine; N6-isopentyl-adenine; 1-methyladenine;1-methylpseudouracil; 1-methylguanine; 2,2-dimethylguanine;2-methyladenine; 2-methylguanine; 3-methylcytosine; 5-methylcytosine;N6-methyladenine; 7-methylguanine; 5-methylaminomethyl uracil; 5-methoxyamino methyl-2-thiouracil; β-D-mannosylqueosine;5-methoxycarbonylmethyluracil; 5-methoxyuracil; 2methylthio-N6-isopentenyladenine; uracil-5-oxyacetic acid methyl ester;pseudouracil; 2-thiocytosine; 5-methyl-2 thiouracil, 2-thiouracil;4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic acid methylester;uracil 5-oxyacetic acid; queosine; 2-thiocytosine; 5-propyluracil;5-propylcytosine; 5-ethyluracil; 5-ethylcytosine; 5-butyluracil;5-phenyluracil; 5-pentylcytosine; and 2,6,-diaminopurine;methylpseudouracil; 1-methylguanine; and 1-methylcytosine.

An oligonucleotide of the invention can also be modified by replacingone or more phosphodiester linkages with alternative linking groups.Alternative linking groups include, but are not limited to embodimentswherein P(O)O is replaced by P(O)S, P(S)S, P(O)NR2, P(O)R, P(O)OR′, CO,or CH2, wherein each R or R′ is independently H or a substituted orunsubstituted C1-C20 alkyl. A preferred set of R substitutions for theP(O)NR2 group are hydrogen and methoxyethyl Linking groups are typicallyattached to each adjacent nucleotide through an —O— bond, but may bemodified to include —N— or —S— bonds. Not all linkages in an oligomerneed to be identical.

The oligonucleotide of the invention can also be modified by conjugationto a polymer, for example, to reduce the rate of excretion whenadministered to an animal. For example, the oligonucleotide can be“PEGylated,” i.e., conjugated to polyethylene glycol (“PEG”). In oneembodiment, the PEG has an average molecular weight ranging from about20 kD to 80 kD. Methods to conjugate an oligonucleotide with a polymer,such PEG, are known to those skilled in the art (See, e.g., Greg T.Hermanson, Bioconjugate Techniques, Academic Press, 1966).

The oligonucleotide of the invention, e.g., a multipartite construct, ananti-C1Q oligonucleotide, a 10.36 oligonucleotide, as described above,or any combination thereof, can be used in the pharmaceuticalcompositions disclosed herein or known in the art.

In one embodiment, the pharmaceutical composition further comprises asolvent.

In one embodiment, the solvent comprises water.

In one embodiment, the solvent comprises a pharmaceutically acceptableorganic solvent. Any useful and pharmaceutically acceptable organicsolvents can be used in the compositions of the invention.

In one embodiment, the pharmaceutical composition is a solution of thesalt in the pharmaceutically acceptable organic solvent.

In one embodiment, the pharmaceutical composition comprises apharmaceutically acceptable organic solvent and further comprises aphospholipid, a sphingomyelin, or phosphatidyl choline. Without wishingto be bound by theory, it is believed that the phospholipid,sphingomyelin, or phosphatidyl choline facilitates formation of aprecipitate when the pharmaceutical composition is injected into waterand can also facilitate controlled release of the oligonucleotide fromthe resulting precipitate. Typically, the phospholipid, sphingomyelin,or phosphatidyl choline is present in an amount ranging from greaterthan 0 to 10 percent by weight of the pharmaceutical composition. In oneembodiment, the phospholipid, sphingomyelin, or phosphatidyl choline ispresent in an amount ranging from about 0.1 to 10 percent by weight ofthe pharmaceutical composition. In one embodiment, the phospholipid,sphingomyelin, or phosphatidyl choline is present in an amount rangingfrom about 1 to 7.5 percent by weight of the pharmaceutical composition.In one embodiment, the phospholipid, sphingomyelin, or phosphatidylcholine is present in an amount ranging from about 1.5 to 5 percent byweight of the pharmaceutical composition. In one embodiment, thephospholipid, sphingomyelin, or phosphatidyl choline is present in anamount ranging from about 2 to 4 percent by weight of the pharmaceuticalcomposition.

The pharmaceutical compositions can optionally comprise one or moreadditional excipients or additives to provide a dosage form suitable foradministration to an animal. When administered to an animal, theoligonucleotide containing pharmaceutical compositions are typicallyadministered as a component of a composition that comprises apharmaceutically acceptable carrier or excipient so as to provide theform for proper administration to the animal. Suitable pharmaceuticalexcipients are described in Remington's Pharmaceutical Sciences1447-1676 (Alfonso R. Gennaro ed., 19th ed. 1995), incorporated hereinby reference. The pharmaceutical compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, pellets, capsules,capsules containing liquids, powders, suppositories, emulsions,aerosols, sprays, suspensions, or any other form suitable for use.

In one embodiment, the pharmaceutical compositions are formulated forintravenous or parenteral administration. Typically, compositions forintravenous or parenteral administration comprise a suitable sterilesolvent, which may be an isotonic aqueous buffer or pharmaceuticallyacceptable organic solvent. Where necessary, the compositions can alsoinclude a solubilizing agent. Compositions for intravenousadministration can optionally include a local anesthetic such aslidocaine to lessen pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Whereoligonucleotide-containing pharmaceutical compositions are to beadministered by infusion, they can be dispensed, for example, with aninfusion bottle containing, for example, sterile pharmaceutical gradewater or saline. Where the pharmaceutical compositions are administeredby injection, an ampoule of sterile water for injection, saline, orother solvent such as a pharmaceutically acceptable organic solvent canbe provided so that the ingredients can be mixed prior toadministration.

In another embodiment, the pharmaceutical compositions are formulated inaccordance with routine procedures as a composition adapted for oraladministration. Compositions for oral delivery can be in the form oftablets, lozenges, aqueous or oily suspensions, granules, powders,emulsions, capsules, syrups, or elixirs, for example. Oral compositionscan include standard excipients such as mannitol, lactose, starch,magnesium stearate, sodium saccharin, cellulose, and magnesiumcarbonate. Typically, the excipients are of pharmaceutical grade. Orallyadministered compositions can also contain one or more agents, forexample, sweetening agents such as fructose, aspartame or saccharin;flavoring agents such as peppermint, oil of wintergreen, or cherry;coloring agents; and preserving agents, to provide a pharmaceuticallypalatable preparation. Moreover, when in tablet or pill form, thecompositions can be coated to delay disintegration and absorption in thegastrointestinal tract thereby providing a sustained action over anextended period of time. Selectively permeable membranes surrounding anosmotically active driving compound are also suitable for orallyadministered compositions. A time-delay material such as glycerolmonostearate or glycerol stearate can also be used.

The pharmaceutical compositions further comprising a solvent canoptionally comprise a suitable amount of a pharmaceutically acceptablepreservative, if desired, so as to provide additional protection againstmicrobial growth. Examples of preservatives useful in the pharmaceuticalcompositions of the invention include, but are not limited to, potassiumsorbate, methylparaben, propylparaben, benzoic acid and its salts, otheresters of parahydroxybenzoic acid such as butylparaben, alcohols such asethyl or benzyl alcohol, phenolic compounds such as phenol, orquaternary compounds such as benzalkonium chlorides (e.g., benzethoniumchloride).

In one embodiment, the pharmaceutical compositions of the inventionoptionally contain a suitable amount of a pharmaceutically acceptablepolymer. The polymer can increase the viscosity of the pharmaceuticalcomposition. Suitable polymers for use in the compositions and methodsof the invention include, but are not limited to,hydroxypropylcellulose, hydoxypropylmethylcellulose (HPMC), chitosan,polyacrylic acid, and polymethacrylic acid.

Typically, the polymer is present in an amount ranging from greater than0 to 10 percent by weight of the pharmaceutical composition. In oneembodiment, the polymer is present in an amount ranging from about 0.1to 10 percent by weight of the pharmaceutical composition. In oneembodiment, the polymer is present in an amount ranging from about 1 to7.5 percent by weight of the pharmaceutical composition. In oneembodiment, the polymer is present in an amount ranging from about 1.5to 5 percent by weight of the pharmaceutical composition. In oneembodiment, the polymer is present in an amount ranging from about 2 to4 percent by weight of the pharmaceutical composition. In oneembodiment, the pharmaceutical compositions of the invention aresubstantially free of polymers.

In one embodiment, any additional components added to the pharmaceuticalcompositions of the invention are designated as GRAS by the FDA for useor consumption by animals. In one embodiment, any additional componentsadded to the pharmaceutical compositions of the invention are designatedas GRAS by the FDA for use or consumption by humans.

The components of the pharmaceutical composition (the solvents and anyother optional components) are preferably biocompatible and non-toxicand, over time, are simply absorbed and/or metabolized by the body.

As described above, the pharmaceutical compositions of the invention canfurther comprise a solvent.

In one embodiment, the solvent comprises water.

In one embodiment, the solvent comprises a pharmaceutically acceptableorganic solvent.

In an embodiment, the oligonucleotide of the invention, e.g., amultipartite construct, an anti-C1Q oligonucleotide, a 10.36oligonucleotide, as described above, or any combination thereof, areavailable as the salt of a metal cation, for example, as the potassiumor sodium salt. These salts, however, may have low solubility in aqueoussolvents and/or organic solvents, typically, less than about 25 mg/mL.The pharmaceutical compositions of the invention comprising (i) an aminoacid ester or amino acid amide and (ii) a protonated oligonucleotide,however, may be significantly more soluble in aqueous solvents and/ororganic solvents. Without wishing to be bound by theory, it is believedthat the amino acid ester or amino acid amide and the protonatedoligonucleotide form a salt, such as illustrated above, and the salt issoluble in aqueous and/or organic solvents.

Similarly, without wishing to be bound by theory, it is believed thatthe pharmaceutical compositions comprising (i) an oligonucleotide of theinvention; (ii) a divalent metal cation; and (iii) optionally acarboxylate, a phospholipid, a phosphatidyl choline, or a sphingomyelinform a salt, such as illustrated above, and the salt is soluble inaqueous and/or organic solvents.

In one embodiment, the concentration of the oligonucleotide of theinvention in the solvent is greater than about 2 percent by weight ofthe pharmaceutical composition. In one embodiment, the concentration ofthe oligonucleotide of the invention in the solvent is greater thanabout 5 percent by weight of the pharmaceutical composition. In oneembodiment, the concentration of the oligonucleotide in the solvent isgreater than about 7.5 percent by weight of the pharmaceuticalcomposition. In one embodiment, the concentration of the oligonucleotidein the solvent is greater than about 10 percent by weight of thepharmaceutical composition. In one embodiment, the concentration of theoligonucleotide in the solvent is greater than about 12 percent byweight of the pharmaceutical composition. In one embodiment, theconcentration of the oligonucleotide in the solvent is greater thanabout 15 percent by weight of the pharmaceutical composition. In oneembodiment, the concentration of the oligonucleotide in the solvent isranges from about 2 percent to 5 percent by weight of the pharmaceuticalcomposition. In one embodiment, the concentration of the oligonucleotidein the solvent is ranges from about 2 percent to 7.5 percent by weightof the pharmaceutical composition. In one embodiment, the concentrationof the oligonucleotide in the solvent ranges from about 2 percent to 10percent by weight of the pharmaceutical composition. In one embodiment,the concentration of the oligonucleotide in the solvent is ranges fromabout 2 percent to 12 percent by weight of the pharmaceuticalcomposition. In one embodiment, the concentration of the oligonucleotidein the solvent is ranges from about 2 percent to 15 percent by weight ofthe pharmaceutical composition. In one embodiment, the concentration ofthe oligonucleotide in the solvent is ranges from about 2 percent to 20percent by weight of the pharmaceutical composition.

Any pharmaceutically acceptable organic solvent can be used in thepharmaceutical compositions of the invention. Representative,pharmaceutically acceptable organic solvents include, but are notlimited to, pyrrolidone, N-methyl-2-pyrrolidone, polyethylene glycol,propylene glycol (i.e., 1,3-propylene glycol), glycerol formal,isosorbid dimethyl ether, ethanol, dimethyl sulfoxide, tetraglycol,tetrahydrofurfuryl alcohol, triacetin, propylene carbonate, dimethylacetamide, dimethyl formamide, dimethyl sulfoxide, and combinationsthereof.

In one embodiment, the pharmaceutically acceptable organic solvent is awater soluble solvent. A representative pharmaceutically acceptablewater soluble organic solvents is triacetin.

In one embodiment, the pharmaceutically acceptable organic solvent is awater miscible solvent. Representative pharmaceutically acceptable watermiscible organic solvents include, but are not limited to, glycerolformal, polyethylene glycol, and propylene glycol.

In one embodiment, the pharmaceutically acceptable organic solventcomprises pyrrolidone. In one embodiment, the pharmaceuticallyacceptable organic solvent is pyrrolidone substantially free of anotherorganic solvent.

In one embodiment, the pharmaceutically acceptable organic solventcomprises N-methyl-2-pyrrolidone. In one embodiment, thepharmaceutically acceptable organic solvent is N-methyl-2-pyrrolidonesubstantially free of another organic solvent.

In one embodiment, the pharmaceutically acceptable organic solventcomprises polyethylene glycol. In one embodiment, the pharmaceuticallyacceptable organic solvent is polyethylene glycol substantially free ofanother organic solvent.

In one embodiment, the pharmaceutically acceptable organic solventcomprises propylene glycol. In one embodiment, the pharmaceuticallyacceptable organic solvent is propylene glycol substantially free ofanother organic solvent.

In one embodiment, the pharmaceutically acceptable organic solventcomprises glycerol formal. In one embodiment, the pharmaceuticallyacceptable organic solvent is glycerol formal substantially free ofanother organic solvent.

In one embodiment, the pharmaceutically acceptable organic solventcomprises isosorbid dimethyl ether. In one embodiment, thepharmaceutically acceptable organic solvent is isosorbid dimethyl ethersubstantially free of another organic solvent.

In one embodiment, the pharmaceutically acceptable organic solventcomprises ethanol. In one embodiment, the pharmaceutically acceptableorganic solvent is ethanol substantially free of another organicsolvent.

In one embodiment, the pharmaceutically acceptable organic solventcomprises dimethyl sulfoxide. In one embodiment, the pharmaceuticallyacceptable organic solvent is dimethyl sulfoxide substantially free ofanother organic solvent.

In one embodiment, the pharmaceutically acceptable organic solventcomprises tetraglycol. In one embodiment, the pharmaceuticallyacceptable organic solvent is tetraglycol substantially free of anotherorganic solvent.

In one embodiment, the pharmaceutically acceptable organic solventcomprises tetrahydrofurfuryl alcohol. In one embodiment, thepharmaceutically acceptable organic solvent is tetrahydrofurfurylalcohol substantially free of another organic solvent.

In one embodiment, the pharmaceutically acceptable organic solventcomprises triacetin. In one embodiment, the pharmaceutically acceptableorganic solvent is triacetin substantially free of another organicsolvent.

In one embodiment, the pharmaceutically acceptable organic solventcomprises propylene carbonate. In one embodiment, the pharmaceuticallyacceptable organic solvent is propylene carbonate substantially free ofanother organic solvent.

In one embodiment, the pharmaceutically acceptable organic solventcomprises dimethyl acetamide. In one embodiment, the pharmaceuticallyacceptable organic solvent is dimethyl acetamide substantially free ofanother organic solvent.

In one embodiment, the pharmaceutically acceptable organic solventcomprises dimethyl formamide. In one embodiment, the pharmaceuticallyacceptable organic solvent is dimethyl formamide substantially free ofanother organic solvent.

In one embodiment, the pharmaceutically acceptable organic solventcomprises at least two pharmaceutically acceptable organic solvents.

In one embodiment, the pharmaceutically acceptable organic solventcomprises N-methyl-2-pyrrolidone and glycerol formal. In one embodiment,the pharmaceutically acceptable organic solvent isN-methyl-2-pyrrolidone and glycerol formal. In one embodiment, the ratioof N-methyl-2-pyrrolidone to glycerol formal ranges from about 90:10 to10:90.

In one embodiment, the pharmaceutically acceptable organic solventcomprises propylene glycol and glycerol formal. In one embodiment, thepharmaceutically acceptable organic solvent is propylene glycol andglycerol formal. In one embodiment, the ratio of propylene glycol toglycerol formal ranges from about 90:10 to 10:90.

In one embodiment, the pharmaceutically acceptable organic solvent is asolvent that is recognized as GRAS by the FDA for administration orconsumption by animals. In one embodiment, the pharmaceuticallyacceptable organic solvent is a solvent that is recognized as GRAS bythe FDA for administration or consumption by humans.

In one embodiment, the pharmaceutically acceptable organic solvent issubstantially free of water. In one embodiment, the pharmaceuticallyacceptable organic solvent contains less than about 1 percent by weightof water. In one embodiment, the pharmaceutically acceptable organicsolvent contains less about 0.5 percent by weight of water. In oneembodiment, the pharmaceutically acceptable organic solvent containsless about 0.2 percent by weight of water. Pharmaceutically acceptableorganic solvents that are substantially free of water are advantageoussince they are not conducive to bacterial growth. Accordingly, it istypically not necessary to include a preservative in pharmaceuticalcompositions that are substantially free of water. Another advantage ofpharmaceutical compositions that use a pharmaceutically acceptableorganic solvent, preferably substantially free of water, as the solventis that hydrolysis of the oligonucleotide is minimized. Typically, themore water present in the solvent the more readily the oligonucleotidecan be hydrolyzed. Accordingly, oligonucleotide containingpharmaceutical compositions that use a pharmaceutically acceptableorganic solvent as the solvent can be more stable than oligonucleotidecontaining pharmaceutical compositions that use water as the solvent.

In one embodiment, comprising a pharmaceutically acceptable organicsolvent, the pharmaceutical composition is injectable.

In one embodiment, the injectable pharmaceutical compositions are ofsufficiently low viscosity that they can be easily drawn into a 20 gaugeand needle and then easily expelled from the 20 gauge needle. Typically,the viscosity of the injectable pharmaceutical compositions are lessthan about 1,200 cps. In one embodiment, the viscosity of the injectablepharmaceutical compositions are less than about 1,000 cps. In oneembodiment, the viscosity of the injectable pharmaceutical compositionsare less than about 800 cps. In one embodiment, the viscosity of theinjectable pharmaceutical compositions are less than about 500 cps.Injectable pharmaceutical compositions having a viscosity greater thanabout 1,200 cps and even greater than about 2,000 cps (for example gels)are also within the scope of the invention provided that thecompositions can be expelled through an 18 to 24 gauge needle.

In one embodiment, comprising a pharmaceutically acceptable organicsolvent, the pharmaceutical composition is injectable and does not forma precipitate when injected into water.

In one embodiment, comprising a pharmaceutically acceptable organicsolvent, the pharmaceutical composition is injectable and forms aprecipitate when injected into water. Without wishing to be bound bytheory, it is believed, for pharmaceutical compositions that comprise aprotonated oligonucleotide and an amino acid ester or amide, that thea-amino group of the amino acid ester or amino acid amide is protonatedby the oligonucleotide to form a salt, such as illustrated above, whichis soluble in the pharmaceutically acceptable organic solvent butinsoluble in water. Similarly, when the pharmaceutical compositioncomprises (i) an oligonucleotide; (ii) a divalent metal cation; and(iii) optionally a carboxylate, a phospholipid, a phosphatidyl choline,or a sphingomyelin, it is believed that the components of thecomposition form a salt, such as illustrated above, which is soluble inthe pharmaceutically acceptable organic solvent but insoluble in water.Accordingly, when the pharmaceutical compositions are injected into ananimal, at least a portion of the pharmaceutical compositionprecipitates at the injection site to provide a drug depot. Withoutwishing to be bound by theory, it is believed that when thepharmaceutically compositions are injected into an animal, thepharmaceutically acceptable organic solvent diffuses away from theinjection site and aqueous bodily fluids diffuse towards the injectionsite, resulting in an increase in concentration of water at theinjection site, that causes at least a portion of the composition toprecipitate and form a drug depot. The precipitate can take the form ofa solid, a crystal, a gummy mass, or a gel. The precipitate, however,provides a depot of the oligonucleotide at the injection site thatreleases the oligonucleotide over time. The components of thepharmaceutical composition, i.e., the amino acid ester or amino acidamide, the pharmaceutically acceptable organic solvent, and any othercomponents are biocompatible and non-toxic and, over time, are simplyabsorbed and/or metabolized by the body.

In one embodiment, comprising a pharmaceutically acceptable organicsolvent, the pharmaceutical composition is injectable and formsliposomal or micellar structures when injected into water (typicallyabout 500 μL are injected into about 4 mL of water). The formation ofliposomal or micellar structures are most often formed when thepharmaceutical composition includes a phospholipid. Without wishing tobe bound by theory, it is believed that the oligonucleotide in the formof a salt, which can be a salt formed with an amino acid ester or amideor can be a salt with a divalent metal cation and optionally acarboxylate, a phospholipid, a phosphatidyl choline, or a sphingomyelin,that is trapped within the liposomal or micellar structure. Withoutwishing to be bound by theory, it is believed that when thesepharmaceutically compositions are injected into an animal, the liposomalor micellar structures release the oligonucleotide over time.

In one embodiment, the pharmaceutical composition further comprising apharmaceutically acceptable organic solvent is a suspension of solidparticles in the pharmaceutically acceptable organic solvent. Withoutwishing to be bound by theory, it is believed that the solid particlescomprise a salt formed between the amino acid ester or amino acid amideand the protonated oligonucleotide wherein the acidic phosphate groupsof the oligonucleotide protonates the amino group of the amino acidester or amino acid amide, such as illustrated above, or comprises asalt formed between the oligonucleotide; divalent metal cation; andoptional carboxylate, phospholipid, phosphatidyl choline, orsphingomyelin, as illustrated above. Pharmaceutical compositions thatare suspensions can also form drug depots when injected into an animal.

By varying the lipophilicity and/or molecular weight of the amino acidester or amino acid amide it is possible to vary the properties ofpharmaceutical compositions that include these components and furthercomprise an organic solvent. The lipophilicity and/or molecular weightof the amino acid ester or amino acid amide can be varied by varying theamino acid and/or the alcohol (or amine) used to form the amino acidester (or amino acid amide). For example, the lipophilicity and/ormolecular weight of the amino acid ester can be varied by varying the R1hydrocarbon group of the amino acid ester. Typically, increasing themolecular weight of R1 increase the lipophilicity of the amino acidester. Similarly, the lipophilicity and/or molecular weight of the aminoacid amide can be varied by varying the R3 or R4 groups of the aminoacid amide.

For example, by varying the lipophilicity and/or molecular weight of theamino acid ester or amino acid amide it is possible to vary thesolubility of the oligonucleotide of the invention in water, to vary thesolubility of the oligonucleotide in the organic solvent, vary theviscosity of the pharmaceutical composition comprising a solvent, andvary the ease at which the pharmaceutical composition can be drawn intoa 20 gauge needle and then expelled from the 20 gauge needle.

Furthermore, by varying the lipophilicity and/or molecular weight of theamino acid ester or amino acid amide (i.e., by varying R1 of the aminoacid ester or R3 and R4 of the amino acid amide) it is possible tocontrol whether the pharmaceutical composition that further comprises anorganic solvent will form a precipitate when injected into water.Although different oligonucleotides exhibit different solubility andbehavior, generally the higher the molecular weight of the amino acidester or amino acid amide, the more likely it is that the salt of theprotonated oligonucleotide and the amino acid ester of the amide willform a precipitate when injected into water. Typically, when R1 of theamino acid ester is a hydrocarbon of about C16 or higher thepharmaceutical composition will form a precipitate when injected intowater and when R1 of the amino acid ester is a hydrocarbon of about C12or less the pharmaceutical composition will not form a precipitate wheninjected into water. Indeed, with amino acid esters wherein R1 is ahydrocarbon of about C12 or less, the salt of the protonatedoligonucleotide and the amino acid ester is, in many cases, soluble inwater. Similarly, with amino acid amides, if the combined number ofcarbons in R3 and R4 is 16 or more the pharmaceutical composition willtypically form a precipitate when injected into water and if thecombined number of carbons in R3 and R4 is 12 or less the pharmaceuticalcomposition will not form a precipitate when injected into water.Whether or not a pharmaceutical composition that further comprises apharmaceutically acceptable organic solvent will form a precipitate wheninjected into water can readily be determined by injecting about 0.05 mLof the pharmaceutical composition into about 4 mL of water at about 98°F. and determining how much material is retained on a 0.22 μm filterafter the composition is mixed with water and filtered. Typically, aformulation or composition is considered to be injectable when no morethan 10% of the formulation is retained on the filter. In oneembodiment, no more than 5% of the formulation is retained on thefilter. In one embodiment, no more than 2% of the formulation isretained on the filter. In one embodiment, no more than 1% of theformulation is retained on the filter.

Similarly, in pharmaceutical compositions that comprise a protonatedoligonucleotide and a diester or diamide of aspartic or glutamic acid,it is possible to vary the properties of pharmaceutical compositions byvarying the amount and/or lipophilicity and/or molecular weight of thediester or diamide of aspartic or glutamic acid. Similarly, inpharmaceutical compositions that comprise an oligonucleotide; a divalentmetal cation; and a carboxylate, a phospholipid, a phosphatidyl choline,or a sphingomyelin, it is possible to vary the properties ofpharmaceutical compositions by varying the amount and/or lipophilicityand/or molecular weight of the carboxylate, phospholipid, phosphatidylcholine, or sphingomyelin.

Further, when the pharmaceutical compositions that further comprises anorganic solvent form a depot when administered to an animal, it is alsopossible to vary the rate at which the oligonucleotide is released fromthe drug depot by varying the lipophilicity and/or molecular weight ofthe amino acid ester or amino acid amide. Generally, the more lipophilicthe amino acid ester or amino acid amide, the more slowly theoligonucleotide is released from the depot. Similarly, when thepharmaceutical compositions that further comprises an organic solventand also further comprise a carboxylate, phospholipid, phosphatidylcholine, sphingomyelin, or a diester or diamide of aspartic or glutamicacid and form a depot when administered to an animal, it is possible tovary the rate at which the oligonucleotide is released from the drugdepot by varying the amount and/or lipophilicity and/or molecular weightof the carboxylate, phospholipid, phosphatidyl choline, sphingomyelin,or the diester or diamide of aspartic or glutamic acid.

Release rates from a precipitate can be measured injecting about 50 μLof the pharmaceutical composition into about 4 mL of deionized water ina centrifuge tube. The time that the pharmaceutical composition isinjected into the water is recorded as T=0. After a specified amount oftime, T, the sample is cooled to about −9° C. and spun on a centrifugeat about 13,000 rpm for about 20 min. The resulting supernatant is thenanalyzed by HPLC to determine the amount of oligonucleotide present inthe aqueous solution. The amount of oligonucleotide in the pelletresulting from the centrifugation can also be determined by collectingthe pellet, dissolving the pellet in about 10 μL of methanol, andanalyzing the methanol solution by HPLC to determine the amount ofoligonucleotide in the precipitate. The amount of oligonucleotide in theaqueous solution and the amount of oligonucleotide in the precipitateare determined by comparing the peak area for the HPLC peakcorresponding to the oligonucleotide against a standard curve ofoligonucleotide peak area against concentration of oligonucleotide.Suitable HPLC conditions can be readily determined by one of ordinaryskill in the art.

Methods of Treatment

The pharmaceutical compositions of the invention are useful in humanmedicine and veterinary medicine. Accordingly, the invention furtherrelates to a method of treating or preventing a condition in an animalcomprising administering to the animal an effective amount of thepharmaceutical composition of the invention.

In one embodiment, the invention relates to methods of treating acondition in an animal comprising administering to an animal in needthereof an effective amount of a pharmaceutical composition of theinvention.

In one embodiment, the invention relates to methods of preventing acondition in an animal comprising administering to an animal in needthereof an effective amount of a pharmaceutical composition of theinvention.

Methods of administration include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, oral, sublingual, intracerebral, intravaginal, transdermal,rectal, by inhalation, or topical. The mode of administration is left tothe discretion of the practitioner. In some embodiments, administrationwill result in the release of the oligonucleotide of the invention,e.g., an aptamer, an drug targeting aptamer, a multipartite construct,or any combination thereof, into the bloodstream.

In one embodiment, the method of treating or preventing a condition inan animal comprises administering to the animal in need thereof aneffective amount of an oligonucleotide by parenterally administering thepharmaceutical composition of the invention. In one embodiment, thepharmaceutical compositions are administered by infusion or bolusinjection. In one embodiment, the pharmaceutical composition isadministered subcutaneously.

In one embodiment, the method of treating or preventing a condition inan animal comprises administering to the animal in need thereof aneffective amount of an oligonucleotide by orally administering thepharmaceutical composition of the invention. In one embodiment, thecomposition is in the form of a capsule or tablet.

The pharmaceutical compositions can also be administered by any otherconvenient route, for example, topically, by absorption throughepithelial or mucocutaneous linings (e.g., oral, rectal, and intestinalmucosa, etc.).

The pharmaceutical compositions can be administered systemically orlocally.

The pharmaceutical compositions can be administered together withanother biologically active agent.

In one embodiment, the animal is a mammal.

In one embodiment the animal is a human.

In one embodiment, the animal is a non-human animal.

In one embodiment, the animal is a canine, a feline, an equine, abovine, an ovine, or a porcine.

The effective amount administered to the animal depends on a variety offactors including, but not limited to the type of animal being treated,the condition being treated, the severity of the condition, and thespecific multipartite construct being administered. A treating physiciancan determine an effective amount of the pharmaceutical composition totreat a condition in an animal.

In one embodiment, the multipartite construct can inhibit angiogenesis.In one embodiment, the multipartite construct can inhibit angiogenesisand the disease being treated is cancer. In one embodiment, the aptamercan inhibit angiogenesis and the disease being treated is a solid tumor.

The multipartite construct can be a multipartite construct that inhibitsa neoplastic growth or a cancer. In embodiments, the cancer comprises anacute lymphoblastic leukemia; acute myeloid leukemia; adrenocorticalcarcinoma; AIDS-related cancers; AIDS-related lymphoma; anal cancer;appendix cancer; astrocytomas; atypical teratoid/rhabdoid tumor; basalcell carcinoma; bladder cancer; brain stem glioma; brain tumor(including brain stem glioma, central nervous system atypicalteratoid/rhabdoid tumor, central nervous system embryonal tumors,astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma,medulloblastoma, medulloepithelioma, pineal parenchymal tumors ofintermediate differentiation, supratentorial primitive neuroectodermaltumors and pineoblastoma); breast cancer; bronchial tumors; Burkittlymphoma; cancer of unknown primary site; carcinoid tumor; carcinoma ofunknown primary site; central nervous system atypical teratoid/rhabdoidtumor; central nervous system embryonal tumors; cervical cancer;childhood cancers; chordoma; chronic lymphocytic leukemia; chronicmyelogenous leukemia; chronic myeloproliferative disorders; coloncancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma;endocrine pancreas islet cell tumors; endometrial cancer;ependymoblastoma; ependymoma; esophageal cancer; esthesioneuroblastoma;Ewing sarcoma; extracranial germ cell tumor; extragonadal germ celltumor; extrahepatic bile duct cancer; gallbladder cancer; gastric(stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinalstromal cell tumor; gastrointestinal stromal tumor (GIST); gestationaltrophoblastic tumor; glioma; hairy cell leukemia; head and neck cancer;heart cancer; Hodgkin lymphoma; hypopharyngeal cancer; intraocularmelanoma; islet cell tumors; Kaposi sarcoma; kidney cancer; Langerhanscell histiocytosis; laryngeal cancer; lip cancer; liver cancer;malignant fibrous histiocytoma bone cancer; medulloblastoma;medulloepithelioma; melanoma; Merkel cell carcinoma; Merkel cell skincarcinoma; mesothelioma; metastatic squamous neck cancer with occultprimary; mouth cancer; multiple endocrine neoplasia syndromes; multiplemyeloma; multiple myeloma/plasma cell neoplasm; mycosis fungoides;myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavitycancer; nasopharyngeal cancer; neuroblastoma; Non-Hodgkin lymphoma;nonmelanoma skin cancer; non-small cell lung cancer; oral cancer; oralcavity cancer; oropharyngeal cancer; osteosarcoma; other brain andspinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovariangerm cell tumor; ovarian low malignant potential tumor; pancreaticcancer; papillomatosis; paranasal sinus cancer; parathyroid cancer;pelvic cancer; penile cancer; pharyngeal cancer; pineal parenchymaltumors of intermediate differentiation; pineoblastoma; pituitary tumor;plasma cell neoplasm/multiple myeloma; pleuropulmonary blastoma; primarycentral nervous system (CNS) lymphoma; primary hepatocellular livercancer; prostate cancer; rectal cancer; renal cancer; renal cell(kidney) cancer; renal cell cancer; respiratory tract cancer;retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sézarysyndrome; small cell lung cancer; small intestine cancer; soft tissuesarcoma; squamous cell carcinoma; squamous neck cancer; stomach(gastric) cancer; supratentorial primitive neuroectodermal tumors;T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma;thymoma; thyroid cancer; transitional cell cancer; transitional cellcancer of the renal pelvis and ureter; trophoblastic tumor; uretercancer; urethral cancer; uterine cancer; uterine sarcoma; vaginalcancer; vulvar cancer; Waldenström macroglobulinemia; or Wilm's tumor.The compositions and methods of the invention can be used to treat theseand other cancers.

Oligonucleotide Probe Methods

Nucleic acid sequences fold into secondary and tertiary motifsparticular to their nucleotide sequence. These motifs position thepositive and negative charges on the nucleic acid sequences in locationsthat enable the sequences to bind to specific locations on targetmolecules, including without limitation proteins and other amino acidsequences. These binding sequences are known in the field as aptamers.Due to the trillions of possible unique nucleotide sequences in even arelatively short stretch of nucleotides (e.g., 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39 or 40 nucleotides), a large variety of motifs canbe generated, resulting in aptamers for almost any desired protein orother target.

As described above, aptamers can be created by randomly generatingoligonucleotides of a specific length, typically 20-80 base pairs long,e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79 or 80 base pairs. These randomoligonucleotides are then incubated with the target of interest (e.g.,tissue, cell, microvesicle, protein, etc). After several wash steps, theoligonucleotides that bind to the target are collected and amplified.The amplified aptamers are iteratively added to the target and theprocess is repeated, often 15-20 times. A common version of this processknown to those of skill in the art as the SELEX method.

The end result comprises one or more oligonucleotide probes/aptamerswith high affinity to the target. The invention provides furtherprocessing of such resulting aptamers that can be use to providedesirable characteristics: 1) competitive binding assays to identifyaptamers to a desired epitope; 2) motif analysis to identify highaffinity binding aptamers in silico; and 3) aptamer selection assays toidentify aptamers that can be used to detect a particular disease. Themethods are described in more detail below and further in the Examples.

The invention further contemplates aptamer sequences that are highlyhomologous to the sequences that are discovered by the methods of theinvention. “High homology” typically refers to a homology of 40% orhigher, preferably 60% or higher, 70% or higher, more preferably 80% orhigher, even more preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or higher between a polynucleotide sequence and areference sequence. In an embodiment, the reference sequence comprisesthe sequence of one or more aptamer provided herein. Percent homologies(also referred to as percent identity) are typically carried out betweentwo optimally aligned sequences. Methods of alignment of sequences forcomparison are well-known in the art. Optimal alignment of sequences andcomparison can be conducted, e.g., using the algorithm in “Wilbur andLipman, Proc Natl Acad Sci USA 80: 726-30 (1983)”. Homology calculationscan also be performed using BLAST, which can be found on the NCBI serverat: www.ncbi.nlm.nih.gov/BLAST/ (Altschul S F, et al, Nucleic Acids Res.1997; 25(17):3389-402; Altschul S F, et al, J Mol. Biol. 1990;215(3):403-10). In the case of an isolated polynucleotide which islonger than or equivalent in length to the reference sequence, e.g., asequence identified by the methods herein, the comparison is made withthe full length of the reference sequence. Where the isolatedpolynucleotide is shorter than the reference sequence, e.g., shorterthan a sequence identified by the methods herein, the comparison is madeto a segment of the reference sequence of the same length (excluding anyloop required by the homology calculation).

The invention further contemplates aptamer sequences that are functionalfragments of the sequences that are discovered by the methods of theinvention. In the context of an aptamer sequence, a “functionalfragment” of the aptamer sequence may comprise a subsequence that bindsto the same target as the full length sequence. In some instances, acandidate aptamer sequence is from a member of a library that contains a5′ leader sequences and/or a 3′ tail sequence. Such leader sequences ortail sequences may serve to facilitate primer binding for amplificationor capture, etc. In these embodiments, the functional fragment of thefull length sequence may comprise the subsequence of the candidateaptamer sequence absent the leader and/or tail sequences.

Competitive Antibody Addition

Known aptamer production methods may involve eluting all bound aptamersfrom the target sequence. In some cases, this may not easily identifythe desired aptamer sequence. For example, when trying to replace anantibody in an assay, it may be desirable to only collect aptamers thatbind to the specific epitope of the antibody being replaced. Theinvention provides a method comprising addition of an antibody that isto be replaced to the aptamer/target reaction in order to allow for theselective collection of aptamers which bind to the antibody epitope. Inan embodiment, the method comprises incubating a reaction mixturecomprising randomly generated oligonucleotides with a target ofinterest, removing unbound aptamers from the reaction mixture that donot bind the target, adding an antibody to the reaction mixture thatbinds to that epitope of interest, and collecting the aptamers that aredisplaced by the antibody. The target can be a biological entity such asdisclosed herein, e.g., a protein.

Motif Analysis

In aptamer experiments, multiple aptamer sequences can be identifiedthat bind to a given target. These aptamers will have various bindingaffinities. It can be time consuming and laborious to generatequantities of these many aptamers sufficient to assess the affinities ofeach. To identify large numbers of aptamers with the highest affinitieswithout physically screening large subsets, the invention provides amethod comprising the analysis of the two dimensional structure of oneor more high affinity aptamers to the target of interest. In anembodiment, the method comprises screening the database for aptamersthat have similar two-dimensional structures, or motifs, but notnecessarily similar primary sequences. In an embodiment, the methodcomprises identifying a high affinity aptamer using traditional methodssuch as disclosed herein or known in the art (e.g. surface plasmonresonance binding assay), approximating the two-dimensional structure ofthe high affinity aptamer, and identifying aptamers from a pool ofsequences that are predicted to have a similar two-dimensional structureto the high affinity aptamer. The method thereby provides a pool ofcandidates that also bind the target of interest. The two-dimensionalstructure of an oligo can be predicting using methods known in the art,e.g., via free energy (ΔG) calculations performed using a commerciallyavailable software program such as Vienna or mFold, for example asdescribed in Mathews, D., Sabina, J., Zucker, M. & Turner, H. Expandedsequence dependence of thermodynamic parameters provides robustprediction of RNA secondary structure. J. Mol. Biol. 288, 911-940(1999); Hofacker et al., Monatshefte f. Chemie 125: 167-188 (1994); andHofacker, I. L. Vienna RNA secondary structure server. Nucleic AcidsRes. 31, 3429-3431 (2003), the contents of which are incorporated hereinby reference in their entirety. See FIGS. 2A-2B. The pool of sequencescan be sequenced from a pool of randomly generated aptamer candidatesusing a high-throughput sequencing platform, such as the Ion Torrentplatform from Thermo Fisher Scientific (Waltham, Mass.) orHiSeq/NextSeq/MiSeq platform from Illumina, Inc (San Diego, Calif.).Identifying aptamers from a pool of sequences that are predicted to havea similar two-dimensional structure to the high affinity aptamer maycomprise loading the resulting sequences into the software program ofchoice to identify members of the pool of sequences with similartwo-dimensional structures as the high affinity aptamer. The affinitiesof the pool of sequences can then be determined in situ, e.g., surfaceplasmon resonance binding assay or the like.

Aptamer Subtraction Methods

In order to develop an assay to detect a disease, for example, cancer,one typically screens a large population of known biomarkers from normaland diseased patients in order to identify markers that correlate withdisease. This process works where discriminating markers are alreadydescribed. In order to address this problem, the invention provides amethod comprising subtracting out non-discriminating aptamers from alarge pool of aptamers by incubating them initially with non-targettissue, microvesicles, cells, or other targets of interest. Thenon-target entities can be from a normal/healthy/non-diseased sample.The aptamers that did not bind to the normal non-target entities arethen incubated with diseased entities. The aptamers that bind to thediseased entities but that did not bind the normal entities are thenpossible candidates for an assay to detect the disease. This process isindependent of knowing the existence of a particular marker in thediseased sample.

Subtraction methods can be used to identify aptamers that preferentiallyrecognize a desired population of targets. In an embodiment, thesubtraction method is used to identify aptamers that preferentiallyrecognize target from a diseased target population over a control (e.g.,normal or non-diseased) population. The diseased target population maybe a tissue or a population of cells or microvesicles from a diseasedindividual or individuals, whereas the control population comprisescorresponding tissue, cells or microvesicles from a non-diseasedindividual or individuals. The disease can be a cancer or other diseasedisclosed herein or known in the art. Accordingly, the method providesaptamers that preferentially identify disease targets versus controltargets.

Circulating microvesicles can be isolated from control samples, e.g.,plasma from “normal” individuals that are absent a disease of interest,such as an absence of cancer. Vesicles in the sample are isolated usinga method disclosed herein or as known in the art. For example, vesiclescan be isolated from the plasma by one of the following methods:filtration, ultrafiltration, nanomembrane ultrafiltration, the ExoQuickreagent (System Biosciences, Inc., Mountain View, Calif.),centrifugation, ultracentrifugation, using a molecular crowding reagent(e.g., TEXIS from Life Technologies), polymer precipitation (e.g.,polyethylene glycol (PEG)), affinity isolation, affinity selection,immunoprecipitation, chromatography, size exclusion, or a combination ofany of these methods. The microvesicles isolated in each case will be amixture of vesicle types and will be various sizes althoughultracentrifugation methods may have more tendencies to produceexosomal-sized vesicles. Randomly generated oligonucleotide libraries(e.g., produced as described in the Examples herein) are incubated withthe isolated normal vesicles. The aptamers that do not bind to thesevesicles are isolated, e.g., by precipitating the vesicles (e.g, withPEG) and collecting the supernatant containing the non-binding aptamers.These non-binding aptamers are then contacted with vesicles isolatedfrom diseased patients (e.g., using the same methods as described above)to allow the aptamers to recognize the disease vesicles. Next, aptamersthat are bound to the diseased vesicles are collected. In an embodiment,the vesicles are isolated then lysed using a chaotropic agent (e.g., SDSor a similar detergent), and the aptamers are then captured by runningthe lysis mixture over an affinity column. The affinity column maycomprise streptavidin beads in the case of biotin conjugated aptamerpools. The isolated aptamers are the amplified. The process can thenthen repeated, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 or more times to achieve aptamers having a desiredselectivity for the target.

In one aspect of the invention, an aptamer profile is identified thatcan be used to characterize a biological sample of interest. In anembodiment, a pool of randomly generated oligonucleotides, e.g., atleast 10, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹²,10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹ or at least 10²⁰oligonucleotides, is contacted with a biological component or target ofinterest from a control population. The oligonucleotides that do notbind the biological component or target of interest from the controlpopulation are isolated and then contacted with a biological componentor target of interest from a test population. The oligonucleotides thatbind the biological component or target of interest from the testpopulation are retained. The retained oligonucleotides can be used torepeat the process by contacting the retained oligonucleotides with thebiological component or target of interest from the control population,isolating the retained oligonucleotides that do not bind the biologicalcomponent or target of interest from the control population, and againcontacting these isolated oligonucleotides with the biological componentor target of interest from the test population and isolating the bindingoligonucleotides. The “component” or “target” can be anything that ispresent in sample to which the oligonucleotides are capable of binding(e.g., tissue, cells, microvesicles, polypeptides, peptide, nucleic acidmolecules, carbohydrates, lipids, etc.). The process can be repeated anynumber of desired iterations, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 or more times. The resultingoligonucleotides comprise aptamers that can differentially detect thetest population versus the control. These aptamers provide an aptamerprofile, which comprises a biosignature that is determined using one ormore aptamer, e.g., a biosignature comprising a presence or level of thecomponent or target which is detected using the one or more aptamer.

An exemplary process is illustrated in FIG. 3, which demonstrates themethod to identify aptamer that preferentially recognize cancer exosomesusing exosomes from normal (non-cancer) individuals as a control. In thefigure, exosomes are exemplified but one of skill will appreciate thatother microvesicles can be used in the same manner. The resultingaptamers can provide a profile that can differentially detect the cancerexosomes from the normal exosomes. One of skill will appreciate that thesame steps can be used to derive an aptamer profile to characterize anydisease or condition of interest. The process can also be applied withtissue, cells, or other targets of interest.

In an embodiment, the invention provides an isolated polynucleotide thatencodes a polypeptide, or a fragment thereof, identified by the methodsabove. The invention further provides an isolated polynucleotide havinga nucleotide sequence that is at least 60% identical to the nucleotidesequence identified by the methods above. More preferably, the isolatednucleic acid molecule is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, identical to thenucleotide sequence identified by the methods above. In the case of anisolated polynucleotide which is longer than or equivalent in length tothe reference sequence, e.g., a sequence identified by the methodsabove, the comparison is made with the full length of the referencesequence. Where the isolated polynucleotide is shorter than thereference sequence, e.g., shorter than a sequence identified by themethods above, the comparison is made to a segment of the referencesequence of the same length (excluding any loop required by the homologycalculation).

In a related aspect, the invention provides a method of characterizing abiological phenotype using an aptamer profile. The aptamer profile canbe determined using the method above. The aptamer profile can bedetermined for a test sample and compared to a control aptamer profile.The phenotype may be a disease or disorder such as a cancer.Characterizing the phenotype can include without limitation providing adiagnosis, prognosis, or theranosis. Thus, the aptamer profile canprovide a diagnostic, prognostic and/or theranostic readout for thesubject from whom the test sample is obtained.

In another embodiment, an aptamer profile is determined for a testsample by contacting a pool of aptamer molecules to the test sample,contacting the same pool of aptamers to a control sample, andidentifying one or more aptamer molecules that differentially bind acomponent or target in the test sample but not in the control sample (orvice versa). A “component” or “target” as used in the context of thebiological test sample or control sample can be anything that is presentin sample to which the aptamers are capable of binding (e.g., tissue,cells, microvesicles, polypeptides, peptide, nucleic acid molecules,carbohydrates, lipids, etc.). For example, if a sample is a plasma orserum sample, the aptamer molecules may bind a polypeptide biomarkerthat is solely expressed or differentially expressed (over- orunderexpressed) in a disease state as compared to a non-diseasedsubject. Comparison of the aptamer profile in the test sample ascompared to the control sample may be based on qualitative andquantitative measure of aptamer binding (e.g., binding versus nobinding, or level of binding in test sample versus different level ofbinding in the reference control sample).

In an aspect, the invention provides a method of identifying atarget-specific aptamer profile, comprising contacting a biological testsample with a pool of aptamer molecules, contacting the pool to acontrol biological sample, identifying one or more aptamers that bind toa component in said test sample but not to the control sample, therebyidentifying an aptamer profile for said biological test sample. In anembodiment, a pool of aptamers is selected against a disease sample andcompared to a reference sample, the aptamers in a subset that bind to acomponent(s) in the disease sample but not in the reference sample canbe sequenced using conventional sequencing techniques to identify thesubset that bind, thereby identifying an aptamer profile for theparticular disease sample. In this way, the aptamer profile provides anindividualized platform for detecting disease in other samples that arescreened. Furthermore, by selecting an appropriate reference or controlsample, the aptamer profile can provide a diagnostic, prognostic and/ortheranostic readout for the subject from whom the test sample isobtained.

In a related aspect, the invention provides a method of selecting a poolof aptamers, comprising: (a) contacting a biological control sample witha pool of oligonucleotides; (b) isolating a first subset of the pool ofoligonucleotides that do not bind the biological control sample; (c)contacting the biological test sample with the first subset of the poolof oligonucleotides; and (d) isolating a second subset of the pool ofoligonucleotides that bind the biological test sample, thereby selectingthe pool of aptamers. The pool of oligonucleotides may comprise anynumber of desired sequences, e.g., at least 10, 10², 10³, 10⁴, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸,10¹⁹ or at least 10²⁰ oligonucleotides may be present in the startingpool. Steps (a)-(d) may be repeated to further hone the pool ofaptamers. In an embodiment, these steps are repeated at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or at least 20times.

As described herein, the biological test sample and biological controlsample may comprise tissues, cells, microvesicles, or biomarkers ofinterest. In an embodiment, the biological test sample and optionallybiological control sample comprise a bodily fluid. The bodily fluid maycomprise without limitation peripheral blood, sera, plasma, ascites,urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovialfluid, aqueous humor, amniotic fluid, cerumen, breast milk,broncheoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid,pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair,tears, cyst fluid, pleural fluid, peritoneal fluid, malignant fluid,pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid,menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stoolwater, pancreatic juice, lavage fluids from sinus cavities,bronchopulmonary aspirates or other lavage fluids. The biological testsample and optionally biological control may also comprise a tumorsample, e.g., cells from a tumor or tumor tissue. In other embodiments,the biological test sample and optionally biological control samplecomprise a cell culture medium. In embodiments, the biological testsample comprises a diseased sample and the biological control samplecomprises a non-diseased sample. Accordingly, the pool of aptamers maybe used to provide a diagnostic, prognostic and/or theranostic readoutfor the disease.

As noted, the invention can be used to assess microvesicles.Microvesicles are powerful biomarkers because the vesicles provide onebiological entity that comprises multiple pieces of information. Forexample as described, a vesicle can have multiple surface antigens, eachof which provides complementary information. Consider a cancer markerand a tissue specific marker. If both markers are individually presentin a sample, e.g., both are circulating proteins or nucleic acids, itmay not be ascertainable whether the cancer marker and the tissuespecific marker are derived from the same anatomical locale. However, ifboth the cancer marker and the tissue specific marker are surfaceantigens on a single microvesicle, the vesicle itself links the twomarkers and provides an indication of a disease (via the cancer marker)and origin of the disease (via the tissue specific marker). Furthermore,the vesicle can have any number of surface antigens and also payloadthat can be assessed. Accordingly, the invention provides a method foridentifying binding agents comprising contacting a plurality ofextracellular microvesicles with a randomly generated library of bindingagents, identifying a subset of the library of binding agents that havean affinity to one or more components of the extracellularmicrovesicles. The binding agents may comprise aptamers, antibodies,and/or any other useful type of binding agent disclosed herein or knownin the art.

In a related aspect, the invention provides a method for identifying aplurality of target ligands comprising, (a) contacting a referencemicrovesicle population with a plurality of ligands that are capable ofbinding one or more microvesicle surface markers, (b) isolating aplurality of reference ligands, wherein the plurality of referenceligands comprise a subset of the plurality of ligands that do not havean affinity for the reference microvesicle population; (c) contactingone or more test microvesicle with the plurality of reference ligands;and (d) identifying a subset of ligands from the plurality of referenceligands that form complexes with a surface marker on the one or moretest microvesicle, thereby identifying the plurality of target ligands.The term “ligand” can refer a molecule, or a molecular group, that bindsto another chemical entity to form a larger complex. Accordingly, abinding agent comprises a ligand. The plurality of ligands may compriseaptamers, antibodies and/or other useful binding agents described hereinor known in the art. The process can also be applied to tissue samples.See, e.g., Examples 19-31 herein.

The invention further provides kits comprising one or more reagent tocarry out the methods above. In an embodiment, the one or more reagentcomprises a library of potential binding agents that comprises one ormore of an aptamer, antibody, and other useful binding agents describedherein or known in the art.

Negative and Positive Aptamer Selection

Aptamers can be used in various biological assays, including numeroustypes of assays which rely on a binding agent. For example, aptamers canbe used instead of or along side antibodies in various immunoassayformats, such as sandwich assays, flow cytometry and IHC. The inventionprovides an aptamer screening method that identifies aptamers that donot bind to any surfaces (substrates, tubes, filters, beads, otherantigens, etc.) throughout the assay steps and bind specifically to anantigen of interest. The assay relies on negative selection to removeaptamers that bind non-target antigen components of the final assay. Thenegative selection is followed by positive selection to identifyaptamers that bind the desired antigen.

In an aspect, the invention provides a method of identifying an aptamerspecific to a target of interest, comprising (a) contacting a pool ofcandidate aptamers with one or more assay components, wherein the assaycomponents do not comprise the target of interest; (b) recovering themembers of the pool of candidate aptamers that do not bind to the one ormore assay components in (a); (c) contacting the members of the pool ofcandidate aptamers recovered in (b) with the target of interest in thepresence of one or more confounding target; and (d) recovering acandidate aptamer that binds to the target of interest in step (c),thereby identifying the aptamer specific to the target of interest. Inthe method, steps (a) and (b) provide negative selection to removeaptamers that bind non-target entities. Conversely, steps (c) and (d)provide positive selection by identifying aptamers that bind the targetof interest but not other confounding targets, e.g., other antigens thatmay be present in a biological sample which comprises the target ofinterest. The pool of candidate aptamers may comprise at least 10, 10²,10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵,10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹ or at least 10²⁰ nucleic acid sequences.

In some embodiments, steps (a)-(b) are optional. In other embodiments,steps (a)-(b) are repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or at least 20 times before positiveselection in step (c) is performed. The positive selection can also beperformed in multiple rounds. Steps (c)-(d) can be repeated at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or atleast 20 times before identifying the aptamer specific to the target ofinterest. Multiple rounds may provide improved stringency of selection.

In some embodiments, the one or more assay components contacted with theaptamer pool during negative selection comprise one or more of asubstrate, a bead, a planar array, a column, a tube, a well, or afilter. One of skill will appreciate that the assay components caninclude any substance that may be part of a desired biological assay.

The target of interest can be any appropriate entity that can bedetected when recognized by an aptamer. In an embodiment, the target ofinterest comprises a protein or polypeptide. As used herein, “protein,”“polypeptide” and “peptide” are used interchangeably unless statedotherwise. The target of interest can be a nucleic acid, including DNA,RNA, and various subspecies of any thereof as disclosed herein or knownin the art. The target of interest can comprise a lipid. The target ofinterest can comprise a carbohydrate. The target of interest can also bea complex, e.g., a complex comprising protein, nucleic acids, lipidsand/or carbohydrates. In some embodiments, the target of interestcomprises a tissue, cell, or microvesicle. In such cases, the aptamermay be a binding agent to a surface antigen or disease antigen.

The surface antigen can be a biomarker of a disease or disorder. In suchcases, the aptamer may be used to provide a diagnosis, prognosis ortheranosis of the disease or disorder. For example, the one or moreprotein may comprise one or more of PSMA, PCSA, B7H3, EpCam, ADAM-10,BCNP, EGFR, IL1B, KLK2, MMP7, p53, PBP, SERPINB3, SPDEF, SSX2, and SSX4.These markers can be used detect a prostate cancer. Additional surfaceantigens and disease antigens are provided in Tables 3-4 herein.

The one or more confounding target can be an antigen other than thetarget of interest. For example, a confounding target can be anotherentity that may be present in a sample to be assayed. As a non-limitingexample, consider that the sample to be assessed is a tissue or bloodsample from an individual. The target of interest may be a protein,e.g., a surface antigen, which is present in the sample. In this case, aconfounding target could be selected from any other antigen that islikely to be present in the sample. Accordingly, the positive selectionshould provide candidate aptamers that recognize the target of interestbut have minimal, if any, interactions with the confounding targets. Insome embodiments, the target of interest and the one or more confoundingtarget comprise the same type of biological entity, e.g., all protein,all nucleic acid, all carbohydrate, or all lipids. As a non-limitingexample, the target of interest can be a protein selected from the groupconsisting of SSX4, SSX2, PBP, KLK2, SPDEF, and EpCAM, and the one ormore confounding target comprises the other members of this group. Inother embodiments, the target of interest and the one or moreconfounding target comprise different types of biological entities,e.g., any combination of protein, nucleic acid, carbohydrate, andlipids. The one or more confounding targets may also comprise differenttypes of biological entities, e.g., any combination of protein, nucleicacid, carbohydrate, and lipids.

In an embodiment, the invention provides an isolated polynucleotide, ora fragment thereof, identified by the methods above. The inventionfurther provides an isolated polynucleotide having a nucleotide sequencethat is at least 60% identical to the nucleotide sequence identified bythe methods above. The isolated polynucleotide is also referred to as anaptamer or oligonucleotide probe. More preferably, the isolated nucleicacid molecule is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more, identical to the nucleotidesequence identified by the methods above. In the case of an isolatedpolynucleotide which is longer than or equivalent in length to thereference sequence, e.g., a sequence identified by the methods above,the comparison is made with the full length of the reference sequence.Where the isolated polynucleotide is shorter than the referencesequence, e.g., shorter than a sequence identified by the methods above,the comparison is made to a segment of the reference sequence of thesame length (excluding any loop required by the homology calculation).

In a related aspect, the invention provides a method of selecting agroup of aptamers, comprising: (a) contacting a pool of aptamers to apopulation of microvesicles from a first sample; (b) enriching a subpoolof aptamers that show affinity to the population of microvesicles fromthe first sample; (c) contacting the subpool to a second population ofmicrovesicles from a second sample; and (d) depleting a second subpoolof aptamers that show affinity to the second population of microvesiclesfrom the second sample, thereby selecting the group of aptamers thathave preferential affinity for the population of microvesicles from thefirst sample. The first sample and/or second sample may comprise abiological fluid such as disclosed herein. For example, the biologicalfluid may include without limitation blood, a blood derivative, plasma,serum or urine. The first sample and/or second sample may also bederived from a cell culture.

In another related aspect, the invention provides a method of selectinga group of aptamers, comprising: (a) contacting a pool of aptamers to atissue from a first sample; (b) enriching a subpool of aptamers thatshow affinity to the tissue from the first sample; (c) contacting thesubpool to a second tissue from a second sample; and (d) depleting asecond subpool of aptamers that show affinity to the second tissue fromthe second sample, thereby selecting the group of aptamers that havepreferential affinity for the tissue from the first sample as comparedto the second sample. The first sample and/or second sample may comprisea fixed tissue such as disclosed herein. For example, the fixed tissuemay include FFPE tissue. The first sample and/or second sample maycomprise a tumor sample.

In an embodiment, the first sample comprises a cancer sample and thesecond sample comprises a control sample, such as a non-cancer sample.The first sample and/or and the second sample may each comprise a pooledsample. For example, the first sample and/or second sample can comprisebodily fluid from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more than 100individuals. In such cases, the members of a pool may be chosen torepresent a desired phenotype. In a non-limiting example, the members ofthe first sample pool may be from patients with a cancer and the membersof the second sample pool may be from non-cancer controls. With tissuesamples, the first sample may comprise tissues from differentindividuals, e.g., from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more than 100individuals. As a non-limiting example, the first sample may comprise afixed tissue from each individual.

Steps (a)-(d) can be repeated a desired number of times in order tofurther enrich the pool in aptamers that have preferential affinity forthe target from the first sample. For example, steps (a)-(d) can berepeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 or more than 20 times. The output from step (d) can be used asthe input to repeated step (a). In embodiment, the first sample and/orsecond sample are replaced with a different sample before repeatingsteps (a)-(d). In a non-limiting example, members of a first sample poolmay be from patients with a cancer and members of a second sample poolmay be from non-cancer controls. During subsequent repetitions of steps(a)-(d), the first sample pool may comprise samples from differentcancer patients than in the prior round/s. Similarly, the second samplepool may comprise samples from different controls than in the priorround/s.

In still another related aspect, the invention provides a method ofenriching a plurality of oligonucleotides, comprising: (a) contacting afirst sample with the plurality of oligonucleotides; (b) fractionatingthe first sample contacted in step (a) and recovering members of theplurality of oligonucleotides that fractionated with the first sample;(c) contacting the recovering members of the plurality ofoligonucleotides from step (b) with a second sample; (d) fractionatingthe second sample contacted in step (c) and recovering members of theplurality of oligonucleotides that did not fractionate with the secondsample; (e) contacting the recovering members of the plurality ofoligonucleotides from step (d) with a third sample; and (f)fractionating the third sample contacted in step (a) and recoveringmembers of the plurality of oligonucleotides that fractionated with thethird sample; thereby enriching the plurality of oligonucleotides. Thesamples can be of any appropriate form as described herein, e.g.,tissue, cells, microvesicles, etc. The first and third samples may havea first phenotype while the second sample has a second phenotype. Thus,positive selection occurs for the samples associated with the firstphenotype and negative selection occurs for the samples associated withthe second phenotype. In one non-limiting example of such selectionschemes, the first phenotype comprises biopsy-positive breast cancer andthe second phenotype comprises non-breast cancer (biopsy-negative orhealthy).

In some embodiments, the first phenotype comprises a medical condition,disease or disorder and the second phenotype comprises a healthy stateor a different state of the medical condition, disease or disorder. Thefirst phenotype can be a healthy state and the second phenotypecomprises a medical condition, disease or disorder. The medicalcondition, disease or disorder can be any detectable medical condition,disease or disorder, including without limitation a cancer, apremalignant condition, an inflammatory disease, an immune disease, anautoimmune disease or disorder, a cardiovascular disease or disorder,neurological disease or disorder, infectious disease or pain. Varioustypes of such conditions are disclosed herein. See, e.g., Section“Phenotypes” herein.

Any useful method to isolate microvesicles in whole or in part can beused to fractionate the samples as appropriate. Several usefultechniques are described herein. In an embodiment, the fractionatingcomprises ultracentrifugation in step (b) and polymer precipitation insteps (d) and (f). In other embodiments, polymer precipitation is usedin all steps. The polymer can be polyethylene glycol (PEG). Anyappropriate form of PEG may be used. For example, the PEG may be PEG8000. The PEG may be used at any appropriate concentration. For example,the PEG can be used at a concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% to isolate the microvesicles. Insome embodiments, the PEG is used at a concentration of 6%.

When the sample comprises an FFPE tissue sample, the sample can besubjected to epitope retrieval, also known as antigen retrieval, priorto the enrichment process. Although tissue fixation is useful for thepreservation of tissue morphology, this process can also have a negativeimpact on immuno detection methods. For example, fixation can alterprotein biochemistry such that the epitope of interest is masked and canno longer bind to the primary antibody. Masking of the epitope can becaused by cross-linking of amino acids within the epitope, cross-linkingunrelated peptides at or near an epitope, altering the conformation ofan epitope, or altering the electrostatic charge of the antigen. Epitoperetrieval refers to any technique in which the masking of an epitope isreversed and epitope-recognition is restored. Techniques for epitoperetrieval are known in the art. For example, enzymes includingProteinase K, Trypsin, and Pepsin have been used successfully to restoreepitope binding. Without being bound by theory, the mechanism of actionmay be the cleavage of peptides that may be masking the epitope. Heatingthe sample may also reverse some cross-links and allows for restorationof secondary or tertiary structure of the epitope. Change in pH orcation concentration may also influence epitope availability.

The contacting can be performed in the presence of a competitor, whichmay reduce non-specific binding events. Any useful competitor can beused. In an embodiment, the competitor comprises at least one of salmonsperm DNA, tRNA, dextran sulfate and carboxymethyl dextran. As desired,different competitors or competitor concentrations can be used atdifferent contacting steps.

The method can be repeated to achieve a desired enrichment. In anembodiment, steps (a)-(f) are repeated at least once. These steps can berepeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or more than 20 times as desired. At the same time, each of thecontacting steps can be repeated as desired. In some embodiments, themethod further comprises: (i) repeating steps (a)-(b) at least onceprior to step (c), wherein the recovered members of the plurality ofoligonucleotides that fractionated with the first sample in step (b) areused as the input plurality of oligonucleotides for the repetition ofstep (a); (ii) repeating steps (c)-(d) at least once prior to step (e),wherein the recovered members of the plurality of oligonucleotides thatdid not fractionate with the second sample in step (d) are used as theinput plurality of oligonucleotides for the repetition of step (c);and/or (iii) repeating steps (e)-(f) at least once, wherein therecovered members of the plurality of oligonucleotides that fractionatedwith the third sample in step (f) are used as the input plurality ofoligonucleotides for the repetition of step (e). Repetitions (i)-(iii)can be repeated any desired number of times, e.g., (i)-(iii) can berepeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 or more than 20 times. In an embodiment, (i)-(iii) each comprisethree repetitions.

The method may further comprise identifying the members of the selectedgroup of aptamers or oligonucleotides, e.g., by DNA sequencing. Thesequencing may be performed by Next Generation sequencing as desired andafter or before any desired step in the method.

The method may also comprise identifying the targets of the selectedgroup of aptamers/oligonucleotides. Useful methods to identify suchtargets are disclosed herein. In a non-limiting example, an enrichedoligonucleotide library is contacted with an appropriate sample (e.g.,the first or third sample), the library is cross-linked to the sample,and the library is recovered. Proteins cross-linked with the recoveredlibrary are identified, e.g., by mass spectrometry.

Oligonucleotide Probe Target Identification

The methods and kits above can be used to identify binding agents thatdifferentiate between two target populations. The invention furtherprovides methods of identifying the targets of such binding agents. Forexample, the methods may further comprise identifying a surface markerof a cell or microvesicle that is recognized by the binding agent.

In an embodiment, the invention provides a method of identifying atarget of a binding agent comprising: (a) contacting the binding agentwith the target to bind the target with the binding agent, wherein thetarget comprises a surface antigen of a cell or microvesicle; (b)disrupting the cell or microvesicle under conditions which do notdisrupt the binding of the target with the binding agent; (c) isolatingthe complex between the target and the binding agent; and (d)identifying the target bound by the binding agent. The binding agent canbe a binding agent identified by the methods above, e.g., anoligonucleotide probe, ligand, antibody, or other useful binding agentthat can differentiate between two target populations, e.g., bydifferentiating between biomarkers thereof.

An illustrative schematic for carrying on the method is shown in FIG. 4.The figure shows a binding agent 402, here an oligonucleotide probe oraptamer for purposes of illustration, tethered to a substrate 401. Thebinding agent 402 can be covalently attached to substrate 401. Thebinding agent 402 may also be non-covalently attached. For example,binding agent 402 can comprise a label which can be attracted to thesubstrate, such as a biotin group which can form a complex with anavidin/streptavidin molecule that is covalently attached to thesubstrate. This can allow a complex to be formed between the aptamer andthe microvesicle while in solution, followed by capture of the aptamerusing the biotin label. The binding agent 402 binds to a surface antigen403 of cell or microvesicle 404. In the step signified by arrow (i), thecell or microvesicle 405 is disrupted while leaving the complex betweenthe binding agent 402 and surface antigen 403 intact. Disrupted cell ormicrovesicle 405 is removed, e.g., via washing or buffer exchange, inthe step signified by arrow (ii). In the step signified by arrow (iii),the surface antigen 403 is released from the binding agent 402. Thesurface antigen 403 can be analyzed to determine its identity usingmethods disclosed herein and/or known in the art. The target of themethod can be any useful biological entity associated with a cell ormicrovesicle. For example, the target may comprise a protein, nucleicacid, lipid or carbohydrate, or other biological entity disclosed hereinor known in the art.

In some embodiments of the method, the target is cross-linked to thebinding agent prior disrupting the cell or microvesicle. Without beingbound by theory, this step may assist in maintaining the complex betweenthe binding agent and the target during the disruption process. Anyuseful method of crosslinking disclosed herein or known in the art canbe used. In embodiments, the cross-linking comprises photocrosslinking,an imidoester crosslinker, dimethyl suberimidate, anN-Hydroxysuccinimide-ester crosslinker, bissulfosuccinimidyl suberate(BS3), an aldehyde, acrolein, crotonaldehyde, formaldehyde, acarbodiimide crosslinker, N,N′-dicyclohexylcarbodiimide (DDC),N,N′-diisopropylcarbodiimide (DIC),1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC orEDAC), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC), aSulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(Sulfo-SMCC), a Sulfo-N-hydroxysuccinimidyl-2-(6-[biotinamido]-2-(p-azido benzamido)-hexanoamido)ethyl-1,3′-dithioproprionate (Sulfo-SBED),2-[N2-(4-Azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotin-amidocaproyl)-L-lysinyl]ethylmethanethiosulfonate (Mts-Atf-Biotin; available from Thermo FisherScientific Inc, Rockford Ill.),2-{N2-[N6-(4-Azido-2,3,5,6-tetrafluorobenzoyl-6-amino-caproyl)-N6-(6-biotinamidocaproyl)-L-lysinylamido]}ethylmethanethiosultonate (Mts-Atf-LC-Biotin; available from Thermo FisherScientific Inc), a photoreactive amino acid (e.g., L-Photo-Leucine andL-Photo-Methionine, see, e.g., Suchanek, M., et al. (2005).Photo-leucine and photo-methionine allow identification ofprotein-protein interactions. Nat. Methods 2:261-267), anN-Hydroxysuccinimide (NHS) crosslinker, an NHS-Azide reagent (e.g.,NHS-Azide, NHS-PEG4-Azide, NHS-PEG12-Azide; each available from ThermoFisher Scientific, Inc.), an NHS-Phosphine reagent (e.g., NHS-Phosphine,Sulfo-NHS-Phosphine; each available from Thermo Fisher Scientific,Inc.), or any combination or modification thereof.

A variety of methods can be used to disrupt the cell or microvesicle.For example, the cellular or vesicular membrane can be disrupted usingmechanical forces, chemical agents, or a combination thereof. Inembodiments, disrupting the cell or microvesicle comprises use of one ormore of a detergent, a surfactant, a solvent, an enzyme, or any usefulcombination thereof. The enzyme may comprise one or more of lysozyme,lysostaphin, zymolase, cellulase, mutanoly sin, a glycanase, a protease,and mannase. The detergent or surfactant may comprise one or more of aoctylthioglucoside (OTG), octyl beta-glucoside (OG), a nonionicdetergent, Triton X, Tween 20, a fatty alcohol, a cetyl alcohol, astearyl alcohol, cetostearyl alcohol, an oleyl alcohol, apolyoxyethylene glycol alkyl ether (Brij), octaethylene glycolmonododecyl ether, pentaethylene glycol monododecyl ether, apolyoxypropylene glycol alkyl ether, a glucoside alkyl ether, decylglucoside, lauryl glucoside, octyl glucoside, a polyoxyethylene glycoloctylphenol ethers, a polyoxyethylene glycol alkylphenol ether,nonoxynol-9, a glycerol alkyl ester, glyceryl laurate, a polyoxyethyleneglycol sorbitan alkyl esters, polysorbate, a sorbitan alkyl ester,cocamide MEA, cocamide DEA, dodecyldimethylamine oxide, a blockcopolymers of polyethylene glycol and polypropylene glycol, poloxamers,polyethoxylated tallow amine (POEA), a zwitterionic detergent,3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), alinear alkylbenzene sulfonate (LAS), a alkyl phenol ethoxylate (APE),cocamidopropyl hydroxysultaine, a betaine, cocamidopropyl betaine,lecithin, an ionic detergent, sodium dodecyl sulfate (SDS), cetrimoniumbromide (CTAB), cetyl trimethylammonium chloride (CTAC), octenidinedihydrochloride, cetylpyridinium chloride (CPC), benzalkonium chloride(BAC), benzethonium chloride (BZT), 5-Bromo-5-nitro-1,3-dioxane,dimethyldioctadecylammonium chloride, dioctadecyldimethylammoniumbromide (DODAB), sodium deoxycholate, nonyl phenoxypolyethoxylethanol(Tergitol-type NP-40; NP-40), ammonium lauryl sulfate, sodium laurethsulfate (sodium lauryl ether sulfate (SLES)), sodium myreth sulfate, analkyl carboxylate, sodium stearate, sodium lauroyl sarcosinate, acarboxylate-based fluorosurfactant, perfluorononanoate,perfluorooctanoate (PFOA or PFO), and a biosurfactant. Mechanicalmethods of disruption that can be used comprise without limitationmechanical shear, bead milling, homogenation, microfluidization,sonication, French Press, impingement, a colloid mill, decompression,osmotic shock, thermolysis, freeze-thaw, desiccation, or any combinationthereof.

As shown in FIG. 4, the binding agent may be tethered to a substrate.The binding agent can be tethered before or after the complex betweenthe binding agent and target is formed. The substrate can be any usefulsubstrate such as disclosed herein or known in the art. In anembodiment, the substrate comprises a microsphere. In anotherembodiment, the substrate comprises a planar substrate. In anotherembodiment, the substrate comprises column material. The binding agentcan also be labeled. Isolating the complex between the target and thebinding agent may comprise capturing the binding agent via the label. Asa non-limiting example, the label can be a biotin label. In such cases,the binding agent can be attached to the substrate via abiotin-avidin/streptavidin binding event.

Methods of identifying the target after release from the binding agentwill depend on the type of target of interest. For example, when thetarget comprises a protein, identifying the target may comprise use ofmass spectrometry (MS), peptide mass fingerprinting (PMF; proteinfingerprinting), sequencing, N-terminal amino acid analysis, C-terminalamino acid analysis, Edman degradation, chromatography, electrophoresis,two-dimensional gel electrophoresis (2D gel), antibody array, andimmunoassay. Nucleic acids can be identified by amplification,hybridization or sequencing.

One of skill will appreciate that the method can be used to identify anyappropriate target, including those not associated with a membrane. Forexample, with respect to the FIG. 4, all steps except for the stepsignified by arrow (i) (i.e., disrupting the cell or microvesicle 405),could be performed for a tissue lysate or a circulating target such as aprotein, nucleic acid, lipid, carbohydrate, or combination thereof. Thetarget can be any useful target, including without limitation a tissue,a cell, an organelle, a protein complex, a lipoprotein, a carbohydrate,a microvesicle, a virus, a membrane fragment, a small molecule, a heavymetal, a toxin, a drug, a nucleic acid, mRNA, microRNA, aprotein-nucleic acid complex, and various combinations, fragments and/orcomplexes of any of these.

In an aspect, the invention provides a method of identifying at leastone protein associated with at least one cell or microvesicle in abiological sample, comprising: a) contacting the at least one cell ormicrovesicle with an oligonucleotide probe library, b) isolating atleast one protein bound by at least one member of the oligonucleotideprobe library in step a); and c) identifying the at least one proteinisolated in step b). The isolating can be performed using any usefulmethod such as disclosed herein, e.g., by immunoprecipitation or captureto a substrate. Similarly, the identifying can be performed using anyuseful method such as disclosed herein, including without limitation useof mass spectrometry, 2-D gel electrophoresis or an antibody array.Examples of such methodology are presented herein in Examples 9-11.

The targets identified by the methods of the invention can be detected,e.g., using the oligonucleotide probes of the invention, for variouspurposes as desired. For example, an identified surface antigen can beused to detect a cell or microvesicle displaying such antigen. In anaspect, the invention provides a method of detecting at least one cellor microvesicle in a biological sample comprising contacting thebiological sample with at least one binding agent to at least onesurface antigen and detecting the at least one cell or microvesiclerecognized by the binding agent to the at least one protein. In anembodiment, the at least one surface antigen is selected from Tables 3-4herein. The at least one surface antigen can be selected those disclosedin International Patent Application Nos. PCT/US2009/62880, filed Oct.30, 2009; PCT/US2009/006095, filed Nov. 12, 2009; PCT/US2011/26750,filed Mar. 1, 2011; PCT/US2011/031479, filed Apr. 6, 2011;PCT/US11/48327, filed Aug. 18, 2011; PCT/US2008/71235, filed Jul. 25,2008; PCT/US10/58461, filed Nov. 30, 2010; PCT/US2011/21160, filed Jan.13, 2011; PCT/US2013/030302, filed Mar. 11, 2013; PCT/US12/25741, filedFeb. 17, 2012; PCT/2008/76109, filed Sep. 12, 2008; PCT/US12/42519,filed Jun. 14, 2012; PCT/US12/50030, filed Aug. 8, 2012; PCT/US12/49615,filed Aug. 3, 2012; PCT/US12/41387, filed Jun. 7, 2012;PCT/US2013/072019, filed Nov. 26, 2013; PCT/US2014/039858, filed May 28,2013; PCT/IB2013/003092, filed Oct. 23, 2013; PCT/US13/76611, filed Dec.19, 2013; PCT/US14/53306, filed Aug. 28, 2014; and PCT/US15/62184, filedNov. 23, 2015; PCT/US16/40157, filed Jun. 29, 2016; PCT/US16/44595,filed Jul. 28, 2016; and PCT/US16/21632, filed Mar. 9, 2016; each ofwhich applications is incorporated herein by reference in its entirety.The at least one surface antigen can be a protein in any of Tables 10-17herein. See Example 9. The at least one binding agent may comprise anyuseful binding agent, including without limitation a nucleic acid, DNAmolecule, RNA molecule, antibody, antibody fragment, aptamer, peptoid,zDNA, peptide nucleic acid (PNA), locked nucleic acid (LNA), lectin,peptide, dendrimer, membrane protein labeling agent, chemical compound,or a combination thereof. In some embodiments, the at least one bindingagent comprises at least one oligonucleotide, such as an oligonucleotideprobe as provided herein. The cell can be part of a tissue.

The at least one binding agent can be used to capture and/or detect theat least one cell or microvesicle, which can be a circulating cell ormicrovesicle, including without limitation a microvesicle shed intobodily fluids. Methods of detecting soluble biomarkers and circulatingcells or microvesicles using binding agents are provided herein. See,e.g., FIGS. 2A-B, which figures describe sandwich assay formats. In someembodiments, the at least one binding agent used to capture the at leastone cell or microvesicle is bound to a substrate. Any useful substratecan be used, including without limitation a planar array, a columnmatrix, or a microbead. See, e.g., FIGS. 2A-B. In some embodiments, theat least one binding agent used to detect the at least one cell ormicrovesicle is labeled. Various useful labels are provided herein orknown in the art, including without limitation a magnetic label, afluorescent moiety, an enzyme, a chemiluminescent probe, a metalparticle, a non-metal colloidal particle, a polymeric dye particle, apigment molecule, a pigment particle, an electrochemically activespecies, a semiconductor nanocrystal, a nanoparticle, a quantum dot, agold particle, a fluorophore, or a radioactive label.

In an embodiment, the detecting is used to characterize a phenotype. Thephenotype can be any appropriate phenotype of interest. In someembodiments, the phenotype is a disease or disorder. The characterizingmay comprise providing diagnostic, prognostic and/or theranosticinformation for the disease or disorder. The characterizing may beperformed by comparing a presence or level of the at least one cell ormicrovesicle to a reference. The reference can be selected per thecharacterizing to be performed. For example, when the phenotypecomprises a disease or disorder, the reference may comprise a presenceor level of the at least one microvesicle in a sample from an individualor group of individuals without the disease or disorder. The comparingcan be determining whether the presence or level of the cell ormicrovesicle differs from that of the reference. In some embodiments,the detected cell or microvesicle is found at higher levels in a healthysample as compared to a diseased sample. In another embodiment, thedetected cell or microvesicle is found at higher levels in a diseasedsample as compared to a healthy sample. When multiplex assays areperformed, e.g., using a plurality of binding agents to differentbiomarkers, some antigens may be observed at a higher level in thebiological samples as compared to the reference whereas other antigensmay be observed at a lower level in the biological samples as comparedto the reference.

The method can be used to detect the at least one cell or microvesiclein any appropriate biological sample. For example, the biological samplemay comprise a bodily fluid, tissue sample or cell culture. The bodilyfluid or tissue sample can be from a subject having or suspected ofhaving a medical condition, a disease or a disorder. Thus, the methodcan be used to provide a diagnostic, prognostic, or theranostic read outfor the subject. Any appropriate bodily fluid can be used, includingwithout limitation peripheral blood, sera, plasma, ascites, urine,cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid,aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolarlavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatoryfluid, female ejaculate, sweat, fecal matter, hair oil, tears, cystfluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme,chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginalsecretions, mucosal secretion, stool water, pancreatic juice, lavagefluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavityfluid, or umbilical cord blood.

The method of the invention can be used to detect or characterize anyappropriate disease or disorder of interest, including withoutlimitation Breast Cancer, Alzheimer's disease, bronchial asthma,Transitional cell carcinoma of the bladder, Giant cellularosteoblastoclastoma, Brain Tumor, Colorectal adenocarcinoma, Chronicobstructive pulmonary disease (COPD), Squamous cell carcinoma of thecervix, acute myocardial infarction (AMI)/acute heart failure, Chron'sDisease, diabetes mellitus type II, Esophageal carcinoma, Squamous cellcarcinoma of the larynx, Acute and chronic leukemia of the bone marrow,Lung carcinoma, Malignant lymphoma, Multiple Sclerosis, Ovariancarcinoma, Parkinson disease, Prostate adenocarcinoma, psoriasis,Rheumatoid Arthritis, Renal cell carcinoma, Squamous cell carcinoma ofskin, Adenocarcinoma of the stomach, carcinoma of the thyroid gland,Testicular cancer, ulcerative colitis, or Uterine adenocarcinoma.

In some embodiments, the disease or disorder comprises a cancer, apremalignant condition, an inflammatory disease, an immune disease, anautoimmune disease or disorder, a cardiovascular disease or disorder,neurological disease or disorder, infectious disease or pain. The cancercan include without limitation one of acute lymphoblastic leukemia;acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancers;AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas;atypical teratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer;brain stem glioma; brain tumor (including brain stem glioma, centralnervous system atypical teratoid/rhabdoid tumor, central nervous systemembryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma,ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymaltumors of intermediate differentiation, supratentorial primitiveneuroectodermal tumors and pineoblastoma); breast cancer; bronchialtumors; Burkitt lymphoma; cancer of unknown primary site; carcinoidtumor; carcinoma of unknown primary site; central nervous systematypical teratoid/rhabdoid tumor; central nervous system embryonaltumors; cervical cancer; childhood cancers; chordoma; chroniclymphocytic leukemia; chronic myelogenous leukemia; chronicmyeloproliferative disorders; colon cancer; colorectal cancer;craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas isletcell tumors; endometrial cancer; ependymoblastoma; ependymoma;esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranialgerm cell tumor; extragonadal germ cell tumor; extrahepatic bile ductcancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinalcarcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinalstromal tumor (GIST); gestational trophoblastic tumor; glioma; hairycell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma;hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposisarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer;lip cancer; liver cancer; lung cancer; malignant fibrous histiocytomabone cancer; medulloblastoma; medulloepithelioma; melanoma; Merkel cellcarcinoma; Merkel cell skin carcinoma; mesothelioma; metastatic squamousneck cancer with occult primary; mouth cancer; multiple endocrineneoplasia syndromes; multiple myeloma; multiple myeloma/plasma cellneoplasm; mycosis fungoides; myelodysplastic syndromes;myeloproliferative neoplasms; nasal cavity cancer; nasopharyngealcancer; neuroblastoma; Non-Hodgkin lymphoma; nonmelanoma skin cancer;non-small cell lung cancer; oral cancer; oral cavity cancer;oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors;ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor;ovarian low malignant potential tumor; pancreatic cancer;papillomatosis; paranasal sinus cancer; parathyroid cancer; pelviccancer; penile cancer; pharyngeal cancer; pineal parenchymal tumors ofintermediate differentiation; pineoblastoma; pituitary tumor; plasmacell neoplasm/multiple myeloma; pleuropulmonary blastoma; primarycentral nervous system (CNS) lymphoma; primary hepatocellular livercancer; prostate cancer; rectal cancer; renal cancer; renal cell(kidney) cancer; renal cell cancer; respiratory tract cancer;retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sézarysyndrome; small cell lung cancer; small intestine cancer; soft tissuesarcoma; squamous cell carcinoma; squamous neck cancer; stomach(gastric) cancer; supratentorial primitive neuroectodermal tumors;T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma;thymoma; thyroid cancer; transitional cell cancer; transitional cellcancer of the renal pelvis and ureter; trophoblastic tumor; uretercancer; urethral cancer; uterine cancer; uterine sarcoma; vaginalcancer; vulvar cancer; Waldenström macroglobulinemia; or Wilm's tumor.The premalignant condition can include without limitation Barrett'sEsophagus. The autoimmune disease can include without limitation one ofinflammatory bowel disease (IBD), Crohn's disease (CD), ulcerativecolitis (UC), pelvic inflammation, vasculitis, psoriasis, diabetes,autoimmune hepatitis, multiple sclerosis, myasthenia gravis, Type Idiabetes, rheumatoid arthritis, psoriasis, systemic lupus erythematosis(SLE), Hashimoto's Thyroiditis, Grave's disease, Ankylosing SpondylitisSjogrens Disease, CREST syndrome, Scleroderma, Rheumatic Disease, organrejection, Primary Sclerosing Cholangitis, or sepsis. The cardiovasculardisease can include without limitation one of atherosclerosis,congestive heart failure, vulnerable plaque, stroke, ischemia, highblood pressure, stenosis, vessel occlusion or a thrombotic event. Theneurological disease can include without limitation one of MultipleSclerosis (MS), Parkinson's Disease (PD), Alzheimer's Disease (AD),schizophrenia, bipolar disorder, depression, autism, Prion Disease,Pick's disease, dementia, Huntington disease (HD), Down's syndrome,cerebrovascular disease, Rasmussen's encephalitis, viral meningitis,neurospsychiatric systemic lupus erythematosus (NPSLE), amyotrophiclateral sclerosis, Creutzfeldt-Jacob disease,Gerstmann-Straussler-Scheinker disease, transmissible spongiformencephalopathy, ischemic reperfusion damage (e.g. stroke), brain trauma,microbial infection, or chronic fatigue syndrome. The pain can includewithout limitation one of fibromyalgia, chronic neuropathic pain, orperipheral neuropathic pain. The infectious disease can include withoutlimitation one of a bacterial infection, viral infection, yeastinfection, Whipple's Disease, Prion Disease, cirrhosis,methicillin-resistant Staphylococcus aureus, HIV, HCV, hepatitis,syphilis, meningitis, malaria, tuberculosis, or influenza. One of skillwill appreciate that oligonucleotide probes or plurality ofoligonucleotides or methods of the invention can be used to assess anynumber of these or other related diseases and disorders.

In a related aspect, the invention provides a kit comprising a reagentfor carrying out the methods herein. In still another related aspect,the invention provides for use of a reagent for carrying out themethods. The reagent may comprise at least one binding agent to the atleast one protein. The binding agent may be an oligonucleotide probe asprovided herein.

Sample Characterization

The oligonucleotide probe/aptamers of the invention can be used tocharacterize a biological sample. For example, an oligonucleotide probeor oligonucleotide probe library can be used to provide a biosignaturefor the sample. The biosignature can indicate a characteristic of thesample, such as a diagnosis, prognosis or theranosis of a disease ordisorder associated with the sample. In some embodiments, thebiosignature comprises a presence or level of one or more biomarkerpresent in the sample. In some embodiments, biosignature comprises apresence or level of the oligonucleotide probe or members of theoligonucleotide probe library that associated with the sample (e.g., byforming a complex with the sample).

In an aspect, the invention provides an aptamer comprising a nucleicacid sequence that is at least about 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 96, 97, 98, 99 or 100 percent homologous to any one of SEQ ID NOs.1-206506; or a functional variation or fragment of any precedingsequence. A functional variation or fragment includes a sequencecomprising modifications that is still capable of binding a targetmolecule, wherein the modifications comprise without limitation at leastone of a deletion, insertion, point mutation, truncation or chemicalmodification. In a related aspect, the invention provides a method ofcharacterizing a disease or disorder, comprising: (a) contacting abiological test sample with one or more aptamer of the invention, e.g.,any of those in this paragraph or modifications thereof; (b) detecting apresence or level of a complex between the one or more aptamer and thetarget bound by the one or more aptamer in the biological test sampleformed in step (a); (c) contacting a biological control sample with theone or more aptamer; (d) detecting a presence or level of a complexbetween the one or more aptamer and the target bound by the one or moreaptamer in the biological control sample formed in step (c); and (e)comparing the presence or level detected in steps (b) and (d), therebycharacterizing the disease or disorder.

The biological test sample and biological control sample can eachcomprise a tissue sample, a cell culture, or a biological fluid. In someembodiments, the biological test sample and biological control samplecomprise the same sample type, e.g., both the test and control samplesare tissue samples or both are fluid samples. In other embodiments,different sample types may be used for the test and control samples. Forexample, the control sample may comprise an engineered or otherwiseartificial sample. In some embodiments, the tissue samples comprisefixed samples.

The biological fluid may comprise a bodily fluid. The bodily fluid mayinclude without limitation one or more of peripheral blood, sera,plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bonemarrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breastmilk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper'sfluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter,hair, tears, cyst fluid, pleural and peritoneal fluid, pericardialfluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus,sebum, vomit, vaginal secretions, mucosal secretion, stool water,pancreatic juice, lavage fluids from sinus cavities, bronchopulmonaryaspirates, blastocyl cavity fluid, or umbilical cord blood. In someembodiments, the bodily fluid comprises blood, serum or plasma.

The biological fluid may comprise microvesicles. For example, thebiological fluid can be a tissue, cell culture, or bodily fluid whichcomprises microvesicles released from cells in the sample. Themicrovesicles can be circulating microvesicles. The biological fluid maycomprise cells. For example, the biological fluid can be a tissue, cellculture, or bodily fluid which comprises cells circulating in thesample.

The one or more aptamer can bind a target biomarker, e.g., a biomarkeruseful in characterizing the sample. The biomarker may comprise apolypeptide or fragment thereof, or other useful biomarker describedherein or known in the art (lipid, carbohydrate, complex, nucleic acid,etc). In embodiments, the polypeptide or fragment thereof is soluble ormembrane bound. Membrane bound polypeptides may comprise a cellularsurface antigen or a microvesicle surface antigen. The biomarker can bea biomarker selected from Table 3 or Table 4. The biomarker can beselected from one of International Patent Application Nos.PCT/US2009/62880, filed Oct. 30, 2009; PCT/US2009/006095, filed Nov. 12,2009; PCT/US2011/26750, filed Mar. 1, 2011; PCT/US2011/031479, filedApr. 6, 2011; PCT/US11/48327, filed Aug. 18, 2011; PCT/US2008/71235,filed Jul. 25, 2008; PCT/US10/58461, filed Nov. 30, 2010;PCT/US2011/21160, filed Jan. 13, 2011; PCT/US2013/030302, filed Mar. 11,2013; PCT/US12/25741, filed Feb. 17, 2012; PCT/2008/76109, filed Sep.12, 2008; PCT/US12/42519, filed Jun. 14, 2012; PCT/US12/50030, filedAug. 8, 2012; PCT/US12/49615, filed Aug. 3, 2012; PCT/US12/41387, filedJun. 7, 2012; PCT/US2013/072019, filed Nov. 26, 2013; PCT/US2014/039858,filed May 28, 2013; PCT/IB2013/003092, filed Oct. 23, 2013;PCT/US13/76611, filed Dec. 19, 2013; PCT/US14/53306, filed Aug. 28,2014; and PCT/US15/62184, filed Nov. 23, 2015; PCT/US16/40157, filedJun. 29, 2016; PCT/US16/44595, filed Jul. 28, 2016; and PCT/US16/21632,filed Mar. 9, 2016; each of which applications is incorporated herein byreference in its entirety.

The characterizing can comprises a diagnosis, prognosis or theranosis ofthe disease or disorder. Various diseases and disorders can becharacterized using the compositions and methods of the invention,including without limitation a cancer, a premalignant condition, aninflammatory disease, an immune disease, an autoimmune disease ordisorder, a cardiovascular disease or disorder, a neurological diseaseor disorder, an infectious disease, and/or pain. See, e.g., sectionherein “Phenotypes” for further details. In embodiments, the disease ordisorder comprises a proliferative or neoplastic disease or disorder.For example, the disease or disorder can be a cancer. In someembodiments, the cancer comprises a breast cancer, ovarian cancer,prostate cancer, lung cancer, colorectal cancer, melanoma, pancreaticcancer, kidney cancer, or brain cancer.

FIG. 10A is a schematic 1000 showing an assay configuration that can beused to detect and/or quantify a target of interest using one or moreoligonucleotide probe of the invention. Capture aptamer 1002 is attachedto substrate 1001. The substrate can be a planar substrate, well,microbead, or other useful substrate as disclosed herein or known in theart. Target of interest 1003 is bound by capture aptamer 1002. Thetarget of interest can be any appropriate entity that can be detectedwhen recognized by an aptamer or other binding agent. The target ofinterest may comprise a protein or polypeptide, a nucleic acid,including DNA, RNA, and various subspecies thereof, a lipid, acarbohydrate, a complex, e.g., a complex comprising protein, nucleicacids, lipids and/or carbohydrates. In some embodiments, the target ofinterest comprises a tissue, cell or microvesicle. The target ofinterest can be a cellular surface antigen or microvesicle surfaceantigen. The target of interest may be a biomarker, e.g., as disclosedherein. The target of interest can be isolated from a sample usingvarious techniques as described herein, e.g., chromatography,filtration, centrifugation, flow cytometry, affinity capture (e.g., to aplanar surface, column or bead), and/or using microfluidics. Detectionaptamer 1004 is also bound to target of interest 1003. Detection aptamer1004 carries label 1005 which can be detected to identify targetcaptured to substrate 1001 via capture aptamer 1002. The label can be afluorescent, radiolabel, enzyme, or other detectable label as disclosedherein. Either capture aptamer 1002 or detection aptamer 1004 can besubstituted with another binding agent, e.g., an antibody. For example,the target may be captured with an antibody and detected with anaptamer, or vice versa. When the target of interest comprises a complex,the capture and detection agents (aptamer, antibody, etc) can recognizethe same or different targets. For example, when the target is a cell ormicrovesicle, the capture agent may recognize one surface antigen whilethe detection agent recognizes microvesicle surface antigen.Alternately, the capture and detection agents can recognize the samesurface antigen.

The aptamers of the invention may be identified and/or used for variouspurposes in the form of DNA or RNA. Unless otherwise specified, one ofskill in the art will appreciate that an aptamer may generally besynthesized in various forms of nucleic acid. The aptamers may alsocarry various chemical modifications and remain within the scope of theinvention.

In some embodiments, an aptamer of the invention is modified to compriseat least one chemical modification. The modification may include withoutlimitation a chemical substitution at a sugar position; a chemicalsubstitution at a phosphate position; and a chemical substitution at abase position of the nucleic acid. In some embodiments, the modificationis selected from the group consisting of: biotinylation, incorporationof a fluorescent label, incorporation of a modified nucleotide, a2′-modified pyrimidine, 3′ capping, conjugation to an amine linker,conjugation to a high molecular weight, non-immunogenic compound,conjugation to a lipophilic compound, conjugation to a drug, conjugationto a cytotoxic moiety, and labeling with a radioisotope, or othermodification as disclosed herein. The position of the modification canbe varied as desired. For example, the biotinylation, fluorescent label,or cytotoxic moiety can be conjugated to the 5′ end of the aptamer. Thebiotinylation, fluorescent label, or cytotoxic moiety can also beconjugated to the 3′ end of the aptamer.

In some embodiments, the cytotoxic moiety is encapsulated in ananoparticle. The nanoparticle can be selected from the group consistingof: liposomes, dendrimers, and comb polymers. In other embodiments, thecytotoxic moiety comprises a small molecule cytotoxic moiety. The smallmolecule cytotoxic moiety can include without limitation vinblastinehydrazide, calicheamicin, vinca alkaloid, a cryptophycin, a tubulysin,dolastatin-10, dolastatin-15, auristatin E, rhizoxin, epothilone B,epithilone D, taxoids, maytansinoids and any variants and derivativesthereof. In still other embodiments, the cytotoxic moiety comprises aprotein toxin. For example, the protein toxin can be selected from thegroup consisting of diphtheria toxin, ricin, abrin, gelonin, andPseudomonas exotoxin A. Non-immunogenic, high molecular weight compoundsfor use with the invention include polyalkylene glycols, e.g.,polyethylene glycol. Appropriate radioisotopes include yttrium-90,indium-111, iodine-131, lutetium-177, copper-67, rhenium-186,rhenium-188, bismuth-212, bismuth-213, astatine-211, and actinium-225.The aptamer may be labeled with a gamma-emitting radioisotope.

In some embodiments of the invention, an active agent is conjugated tothe aptamer. For example, the active agent may be a therapeutic agent ora diagnostic agent. The therapeutic agent may be selected from the groupconsisting of tyrosine kinase inhibitors, kinase inhibitors,biologically active agents, biological molecules, radionuclides,adriamycin, ansamycin antibiotics, asparaginase, bleomycin, busulphan,cisplatin, carboplatin, carmustine, capecotabine, chlorambucil,cytarabine, cyclophosphamide, camptothecin, dacarbazine, dactinomycin,daunorubicin, dexrazoxane, docetaxel, doxorubicin, etoposide,epothilones, floxuridine, fludarabine, fluorouracil, gemcitabine,hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine,mechlorethamine, mercaptopurine, melphalan, methotrexate, rapamycin(sirolimus), mitomycin, mitotane, mitoxantrone, nitrosurea, paclitaxel,pamidronate, pentostatin, plicamycin, procarbazine, rituximab,streptozocin, teniposide, thioguanine, thiotepa, taxanes, vinblastine,vincristine, vinorelbine, taxol, combretastatins, discodermolides,transplatinum, anti-vascular endothelial growth factor compounds(“anti-VEGFs”), anti-epidermal growth factor receptor compounds(“anti-EGFRs”), 5-fluorouracil and derivatives, radionuclides,polypeptide toxins, apoptosis inducers, therapy sensitizers, enzyme oractive fragment thereof, and combinations thereof.

Oligonucleotide Pools to Characterize a Sample

The complexity and heterogeneity present in biology challenges theunderstanding of biological systems and disease. Diversity exists atvarious levels, e.g., within and between cells, tissues, individuals anddisease states. See, e.g., FIG. 11A. FIG. 11B overviews variousbiological entities that can be assessed to characterize such samples.As shown in FIG. 11B, as one moves from assessing DNA, to RNA, toprotein, and finally to protein complexes, the amount of diversity andcomplexity increases dramatically. The oligonucleotide probe librarymethod of the invention can be used characterize complex biologicalsources, e.g., tissue samples, cells, circulating tumor cells,microvesicles, and complexes such as protein and proteolipid complexes.

Current methods to characterize biological samples may not adequatelyaddress such complexity and diversity. As shown in FIG. 11C, suchcurrent methods often have a trade off between measuring diversity andcomplexity. As an example, consider high throughput sequencingtechnology. Next generation approaches may query many 1000s of moleculartargets in a single assay. However, such approaches only probeindividual DNA and/or RNA molecules, and thus miss out on the greatdiversity of proteins and biological complexes. On the other hand, flowcytometry can probe biological complexes, but are limited to a smallnumber of pre-defined ligands. For example, a single assay can probe ahandful of differentially labeled antibodies to pre-defined targets.

The oligonucleotide probe libraries of the invention address the abovechallenges. The size of the starting library can be adjusted to measureas many different entities as there are library members. For example,the initial untrained oligonucleotide library has the potential tomeasure 10¹² or more biological features. A larger and/or differentlibrary can be constructed as desired. The technology is adapted to finddifferences between samples without assumptions about what “should bedifferent.” For example, the probe library may distinguish based onindividual proteins, protein modifications, protein complexes, lipids,nucleic acids, different folds or conformations, or whatever is therethat distinguishes a sample of interest. Thus, the method provides anunbiased approach to identify differences in biological samples that canbe used to identify different populations of interest.

In the context herein, the use of the oligonucleotide library probe toassess a sample may be referred to as Adaptive Dynamic ArtificialPoly-ligand Targeting, or ADAPT™ (alternately referred to as TopologicalOligonucleotide Profiling: TOP™). Although as noted the terms aptamerand oligonucleotides are typically used interchangeable herein, somedifferences between “classic” individual aptamers and ADAPT probes areas follows. Individual aptamers may comprise individual oligonucleotidesselected to bind to a known specific target in an antibody-like“key-in-lock” binding mode. They may be evaluated individually based onspecificity and binding affinity to the intended target. However, ADAPTprobes may comprise a library of oligonucleotides intended to producemulti-probe signatures. The ADAPT probes comprise numerous potentialbinding modalities (electrostatic, hydrophobic, Watson-Crick,multi-oligo complexes, etc.). The ADAPT probe signatures have thepotential to identify heterogeneous patient subpopulations. For example,a single ADAPT library can be assembled to differentiate multiplebiological states. Unlike classic single aptamers, the binding targetsmay or may not be isolated or identified. It will be understood thatscreening methods that identify individual aptamers, e.g., SELEX, canalso be used to enrich a naïve library of oligonucleotides to identify aADAPT probe library.

The general method of the invention is outlined in FIG. 11D. One inputto the method comprises a randomized oligonucleotide library with thepotential to measure 10¹² or more biological features. As outlined inthe figure, the method identifies a desired number (e.g., ˜10⁵-10⁶) thatare different between two input sample types. The randomizedoligonucleotide library is contacted with a first and a second sampletype, and oligonucleotides that bind to each sample are identified. Thebound oligonucleotide populations are compared and oligonucleotides thatspecifically bind to one or the other biological input sample areretained for the oligonucleotide probe library, whereas oligonucleotidesthat bind both biological input samples are discarded. This trainedoligonucleotide probe library can then be contacted with a new testsample and the identities of oligonucleotides that bind the test sampleare determined. The test sample is characterized based on the profile ofoligonucleotides that bound. See, e.g., FIG. 11H.

Extracellular vesicles provide an attractive vehicle to profile thebiological complexity and diversity driven by many inter-relatedsources. There can be a great deal of heterogeneity betweenpatient-to-patient microvesicle populations, or even in microvesiclepopulations from a single patient under different conditions (e.g.,stress, diet, exercise, rest, disease, etc). Diversity of molecularphenotypes within microvesicle populations in various disease states,even after microvesicle isolation and sorting by vesicle biomarkers, canpresent challenges identifying surface binding ligands. This situationis further complicated by vesicle surface-membrane protein complexes.The oligonucleotide probe library can be used to address such challengesand allow for characterization of biological phenotypes. The approachcombines the power of diverse oligonucleotide libraries and highthroughput (next-generation) sequencing technologies to probe thecomplexity of extracellular microvesicles. See FIG. 11E.

ADAPT™ profiling may provide quantitative measurements of dynamic eventsin addition to detection of presence/absence of various biomarkers in asample. For example, the binding probes may detect protein complexes orother post-translation modifications, allowing for differentiation ofsamples with the same proteins but in different biologicalconfigurations. Such configurations are illustrated in FIGS. 11F-G. InFIG. 11F, microvesicles with various surface markers are shown from anexample microvesicle sample population: Sample Population A. Theindicated Bound Probing Oligonucleotides 1101 are contacted to twosurface markers 1102 and 1103 in a given special relationship. Here,probes unique to these functional complexes and spatial relationshipsmay be retained. In contrast, in microvesicle Sample Population B shownin FIG. 11F, the two surface markers 1102 and 1103 are found indisparate spacial relationship. Here, probes 1101 are not bound due toabsence of the spatial relationship of the interacting components 1102and 1103.

An illustrative approach 1110 for using ADAPT profiling to assess asample is shown in FIG. 11H. The probing library 1111 is mixed withsample 1112. The sample can be as described herein, e.g., a bodily fluidfrom a subject having or suspected of having a disease. The probes areallowed to bind the sample 1120 and the microvesicles are pelleted 1115.The supernatant 1114 comprising unbound oligonucleotides is discarded.Oligonucleotide probes bound to the pellet 1115 are eluted 1116 andsequenced 1117. The profile 1118 generated by the bound oligonucleotideprobes as determined by the sequencing 1117 is used to characterize thesample 1112. For example, the profile 1118 can be compared to areference, e.g., to determine if the profile is similar or differentfrom a reference profile indicative of a disease or healthy state, orother phenotypic characterization of interest. The comparison mayindicate the presence of a disease, provide a diagnosis, prognosis ortheranosis, or otherwise characterize a phenotype associated with thesample 1112. FIG. 11I illustrates another schematic for using ADAPTprofiling to characterize a phenotype. A patient sample such as a bodilyfluid disclosed herein is collected 1121. The sample is contacted withthe ADAPT library pool 1122. Microvesicles (MVs) are isolated from thecontacted sample 1123, e.g., using ultracentrifugation, filtration,polymer precipitation or other appropriate technique or combination oftechniques disclosed herein. Oligonucleotides that bound the isolatedmicrovesicles are collected and identity is determined 1124. Theidentity of the bound oligonucleotides can be determined by any usefultechnique such as sequencing, high throughput sequencing (e.g., NGS),amplification including without limitation qPCR, or hybridization suchas to a planar or particle based array. The identity of the boundoligonucleotides is used to characterize the sample, e.g., as containingdisease related microvesicles.

The approaches outlined in FIG. 11 can be adapted to any desired sampletype, e.g., tissues, cells, microvesicles, circulating biomarkers, andconstituents of any of these.

In an aspect, the invention provides a method of characterizing a sampleby contacting the sample with a pool of different oligonucleotides(which can be referred to as an aptamer pool or oligonucleotide probelibrary), and determining the frequency at which variousoligonucleotides in the pool bind the sample. For example, a pool ofoligonucleotides is identified that preferentially bind to tissues,cells or microvesicles from cancer patients as compared to non-cancerpatients. A test sample, e.g., from a patient suspected of having thecancer, is collected and contacted with the pool of oligonucleotides.Oligonucleotides that bind the test sample are eluted from the testsample, collected and identified, and the composition of the boundoligonucleotides is compared to those known to bind cancer samples.Various sequencing, amplification and hybridization techniques can beused to identify the eluted oligonucleotides. For example, when a largepool of oligonucleotides is used, oligonucleotide identification can beperformed by high throughput methods such as next generation sequencingor via hybridization. If the test sample is bound by the oligonucleotidepool in a similar manner (e.g., as determined by bioinformaticsclassification methods) to the sample from cancer patients, then thetest sample is indicative of cancer as well. Using this method, a poolof oligonucleotides that bind one or more antigen can be used tocharacterize the sample without necessarily knowing the precise targetof each member of the pool of oligonucleotides. Thus, the pool ofoligonucleotides provide a biosignature. Examples 5-7 and 9-31 andothers herein illustrate embodiments of the invention.

In an aspect, the invention provides a method for characterizing acondition for a test sample comprising: contacting a sample with aplurality of oligonucleotide capable of binding one or more target(s)present in the sample, identifying a set of oligonucleotides that form acomplex with the sample wherein the set is predetermined to characterizea condition for the sample, thereby characterizing a condition for asample. The sample can be any useful sample such as disclosed herein,e.g., a tissue, cell, microvesicle, or biomarker sample, or any usefulcombination thereof.

In an related aspect, the invention provides a method for identifying aset of oligonucleotides associated with a test sample, comprising: (a)contacting a sample with a plurality of oligonucleotides, isolating aset of oligonucleotides that form a complex with the sample, (b)determining sequence and/or copy number for each of theoligonucleotides, thereby identifying a set of oligonucleotidesassociated with the test sample. The sample can be any useful samplesuch as disclosed herein, e.g., a tissue, cell, microvesicle, orbiomarker sample, or any useful combination thereof.

In still another related aspect, the invention provides a method ofdiagnosing a sample as cancerous or predisposed to be cancerous,comprising contacting the sample with a plurality of oligonucleotidesthat are predetermined to preferentially form a complex with a cancersample as compared to a non-cancer sample. The sample can be any usefulsample such as disclosed herein, e.g., a tissue, cell, microvesicle, orbiomarker sample, or any useful combination thereof.

The oligonucleotides can be identified by sequencing, e.g., by dyetermination (Sanger) sequencing or high throughput methods. Highthroughput methods can comprise techniques to rapidly sequence a largenumber of nucleic acids, including next generation techniques such asMassively parallel signature sequencing (MPSS; Polony sequencing; 454pyrosequencing; Illumina (Solexa; MiSeq/HiSeq/NextSeq/etc) sequencing;SOLiD sequencing; Ion Torrent semiconductor sequencing; DNA nanoballsequencing; Heliscope single molecule sequencing; Single molecule realtime (SMRT) sequencing, or other methods such as Nanopore DNAsequencing; Tunnelling currents DNA sequencing; Sequencing byhybridization; Sequencing with mass spectrometry; Microfluidic Sangersequencing; Microscopy-based techniques; RNAP sequencing; In vitro virushigh-throughput sequencing. The oligonucleotides may also be identifiedby hybridization techniques. For example, a microarray havingaddressable locals to hybridize and thereby detect the various membersof the pool can be used. Alternately, detection can be based on one ormore differentially labelled oligonucleotides that hybridize withvarious members of the oligonucleotide pool. The detectable signal ofthe label can be associated with a nucleic acid molecule that hybridizeswith a stretch of nucleic acids present in various oligonucleotides. Thestretch can be the same or different as to one or more oligonucleotidesin a library. The detectable signal can comprise fluorescence agents,including color-coded barcodes which are known, such as in U.S. PatentApplication Pub. No. 20140371088, 2013017837, and 20120258870. Otherdetectable labels (metals, radioisotopes, etc) can be used as desired.

The plurality or pool of oligonucleotides can comprise any desirednumber of oligonucleotides to allow characterization of the sample. Invarious embodiments, the pool comprises at least 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80,90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or at least 10000different oligonucleotide members.

The plurality of oligonucleotides can be pre-selected through one ormore steps of positive or negative selection, wherein positive selectioncomprises selection of oligonucleotides against a sample havingsubstantially similar characteristics compared to the test sample, andwherein negative selection comprises selection of oligonucleotidesagainst a sample having substantially different characteristics comparedto the test sample. Substantially similar characteristics mean that thesamples used for positive selection are representative of the testsample in one or more characteristic of interest. For example, thesamples used for positive selection can be from cancer patients or celllines and the test sample can be a sample from a patient having orsuspected to have a cancer. Substantially different characteristics meanthat the samples used for negative selection differ from the test samplein one or more phenotype/characteristic of interest. For example, thesamples used for negative selection can be from individuals or celllines that do not have cancer (e.g., “normal,” “healthy” or otherwise“control” samples) and the test sample can be a sample from a patienthaving or suspected to have a cancer. The cancer can be a breast cancer,ovarian cancer, prostate cancer, lung cancer, colorectal cancer,melanoma, brain cancer, pancreatic cancer, kidney cancer, or othercancer such as disclosed herein.

By selecting samples representative of the desired phenotypes to detectand/or distinguish, the characterizing can comprise a diagnosis,prognosis or theranosis for any number of diseases or disorders. Variousdiseases and disorders can be characterized using the compositions andmethods of the invention, including without limitation a cancer, apremalignant condition, an inflammatory disease, an immune disease, anautoimmune disease or disorder, a cardiovascular disease or disorder, aneurological disease or disorder, an infectious disease, and/or pain.See, e.g., section herein “Phenotypes” for further details. Inembodiments, the disease or disorder comprises a proliferative orneoplastic disease or disorder. For example, the disease or disorder canbe a cancer.

FIG. 10B is a schematic 1010 showing use of an oligonucleotide pool tocharacterize a phenotype of a sample, such as those listed above. A poolof oligonucleotides to a target of interest is provided 1011. Forexample, the pool of oligonucleotides can be enriched to target atissue, cell, microvesicle biomarker, or any combination thereof. Themembers of the pool may bind different targets (e.g., differentproteins) or different epitopes of the same target (e.g., differentepitopes of a single protein). The pool is contacted with a test sampleto be characterized 1012. For example, the test sample may be abiological sample from an individual having or suspected of having agiven disease or disorder. The mixture is washed to remove unboundoligonucleotides. The remaining oligonucleotides are eluted or otherwisedisassociated from the sample and collected 1013. The collectedoligonucleotides are identified, e.g., by sequencing or hybridization1014. The presence and/or copy number of the identified is used tocharacterize the phenotype 1015.

FIG. 10C is a schematic 1020 showing an implementation of the method inFIG. 10B. A pool of oligonucleotides identified as binding amicrovesicle population is provided 1019. The input sample comprises atest sample comprising microvesicles 1022. For example, the test samplemay be a biological sample from an individual having or suspected ofhaving a given disease or disorder. The pool is contacted with theisolated microvesicles to be characterized 1023. The microvesiclepopulation can be isolated before or after the contacting 1023 from thesample using various techniques as described herein, e.g.,chromatography, filtration, ultrafiltration, centrifugation,ultracentrifugation, flow cytometry, affinity capture (e.g., to a planarsurface, column or bead), polymer precipitation, and/or usingmicrofluidics. The mixture is washed to remove unbound oligonucleotidesand the remaining oligonucleotides are eluted or otherwise disassociatedfrom the sample and collected 1024. The collected oligonucleotides areidentified 1025 and the presence and/or copy number of the retainedoligonucleotides is used to characterize the phenotype 1026 as above.

As noted, in embodiment of FIG. 10C, the pool of oligonucleotides 1019is directly contacted with a biological sample that comprises or isexpected to comprise microvesicles. Microvesicles are thereafterisolated from the sample and the mixture is washed to remove unboundoligonucleotides and the remaining oligonucleotides are disassociatedand collected 1024. The following steps are performed as above. As anexample of this alternate configuration, a biological sample, e.g., ablood, serum or plasma sample, is directly contacted with the pool ofoligonucleotides. Microvesicles are then isolated by various techniquesdisclosed herein, including without limitation ultracentrifugation,ultrafiltration, flow cytometry, affinity isolation, polymerprecipitation, chromatography, various combinations thereof, or thelike. Remaining oligonucleotides are then identified, e.g., bysequencing, hybridization or amplification.

In other embodiments, an enriched library of oligonucleotide probes isused to assess a tissue sample. In some embodiments, the pool is used tostain the sample in a manner similar to IHC. Such method may be referredto herein as PHC, or polyligand histochemistry. FIG. 10D provides anoutline 1030 of such method. An aptamer pool is provided that has beenenriched against a tissue of interest 1031. The pool is contacted with atissue sample 1032. The tissue sample can be in a format such asdescribed herein. As a non-limiting example, the tissue sample can be afixed tumor sample. The sample may be a FFPE sample fixed to a glassslide or membrane. The sample is washed to remove unbound members of theaptamer pool and the remaining aptamers are visualized 1033. Anyappropriate method to visualize the aptamers can be used. In an example,the aptamer pool is biotinylated and the bound aptamer are visualizedusing streptavidin-horse radish peroxidase (SA-HRP). As describedherein, other useful visualization methods are known in the art,including alternate labeling. The visualized sample is scored todetermine the amount of staining 1034. For example a pathologist canscore the slide as in IHC. The score can be used to characterize thesample 1035 as described herein. For example, a score of +1 or highermay indicate that the sample is a cancer sample, or is a cancer sampleexpressing a given biomarker. See Examples 19-31 herein.

In a related aspect, the invention provides a composition of mattercomprising a plurality of oligonucleotides that can be used to carry outthe methods comprising use of an oligonucleotide pool to characterize aphenotype. The plurality of oligonucleotides can comprise any of thosedescribed herein.

In an aspect, the invention provides a method for identifyingoligonucleotides specific for a test sample. The method comprises: (a)enriching a plurality of oligonucleotides for a sample to provide a setof oligonucleotides predetermined to form a complex with a targetsample; (b) contacting the plurality in (a) with a test sample to allowformation of complexes of oligonucleotides with test sample; (c)recovering oligonucleotides that formed complexes in (b) to provide arecovered subset of oligonucleotides; and (d) profiling the recoveredsubset of oligonucleotides by high-throughput sequencing, amplificationor hybridization, thereby identifying oligonucleotides specific for atest sample. The test sample may comprise tissue, cells, microvesicles,biomarkers, or other biological entities of interest. Theoligonucleotides may comprise RNA, DNA or both. In some embodiment, themethod further comprises performing informatics analysis to identify asubset of oligonucleotides comprising sequence identity of at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or at least 99% to the oligonucleotides predetermined to forma complex with the target sample.

One of skill will appreciate that the method can be used to identify anyappropriate target. The target can be any useful target, includingwithout limitation a cell, an organelle, a protein complex, alipoprotein, a carbohydrate, a microvesicle, a virus, a membranefragment, a small molecule, a heavy metal, a toxin, a drug, a nucleicacid (including without limitation microRNA (miR) and messenger RNA(mRNA)), a protein-nucleic acid complex, and various combinations,fragments and/or complexes of any of these. The target can, e.g.,comprise a mixture of such biological entities.

In an aspect, the invention also provides a method comprising contactingan oligonucleotide or plurality of oligonucleotides with a sample anddetecting the presence or level of binding of the oligonucleotide orplurality of oligonucleotides to a target in the sample, wherein theoligonucleotide or plurality of oligonucleotides can be those providedby the invention above. The sample may comprise a biological sample, anorganic sample, an inorganic sample, a tissue, a cell culture, a bodilyfluid, blood, serum, a cell, a microvesicle, a protein complex, a lipidcomplex, a carbohydrate, or any combination, fraction or variationthereof. The target may comprise a cell, an organelle, a proteincomplex, a lipoprotein, a carbohydrate, a microvesicle, a membranefragment, a small molecule, a heavy metal, a toxin, or a drug.

In another aspect, the invention provides a method comprising: a)contacting a sample with an oligonucleotide probe library comprising atleast 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶,10¹⁷, or at least 10¹⁸ different oligonucleotide sequencesoligonucleotides to form a mixture in solution, wherein theoligonucleotides are capable of binding a plurality of entities in thesample to form complexes, wherein optionally the oligonucleotide probelibrary comprises an oligonucleotide or plurality of oligonucleotides asprovided by the invention above; b) partitioning the complexes formed instep (a) from the mixture; and c) recovering oligonucleotides present inthe complexes partitioned in step (b) to identify an oligonucleotideprofile for the sample.

In still another aspect, the invention provides a method for generatingan enriched oligonucleotide probe library comprising: a) contacting afirst oligonucleotide library with a biological test sample and abiological control sample, wherein complexes are formed betweenbiological entities present in the biological samples and a plurality ofoligonucleotides present in the first oligonucleotide library; b)partitioning the complexes formed in step (a) and isolating theoligonucleotides in the complexes to produce a subset ofoligonucleotides for each of the biological test sample and biologicalcontrol sample; c) contacting the subsets of oligonucleotides in (b)with the biological test sample and biological control sample whereincomplexes are formed between biological entities present in thebiological samples and a second plurality of oligonucleotides present inthe subsets of oligonucleotides to generate a second subset group ofoligonucleotides; and d) optionally repeating steps b)-c), one, two,three or more times to produce a respective third, fourth, fifth or moresubset group of oligonucleotides, thereby producing the enrichedoligonucleotide probe library. In a related aspect, the inventionprovides a plurality of oligonucleotides comprising at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 300,400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000,90000, 100000, 200000, 300000, 400000, or 500000 differentoligonucleotide sequences, wherein the plurality results from the methodin this paragraph, wherein the library is capable of distinguishing afirst phenotype from a second phenotype. In some embodiments, the firstphenotype comprises a disease or disorder and the second phenotypecomprises a healthy state; or wherein the first phenotype comprises adisease or disorder and the second phenotype comprises a differentdisease or disorder; or wherein the first phenotype comprises a stage orprogression of a disease or disorder and the second phenotype comprisesa different stage or progression of the same disease or disorder; orwherein the first phenotype comprises a positive response to a therapyand the second phenotype comprises a negative response to the sametherapy.

In yet another aspect, the invention provides a method of characterizinga disease or disorder, comprising: a) contacting a biological testsample with the oligonucleotide or plurality of oligonucleotidesprovided by the invention; b) detecting a presence or level of complexesformed in step (a) between the oligonucleotide or plurality ofoligonucleotides provided by the invention and a target in thebiological test sample; and c) comparing the presence or level detectedin step (b) to a reference level from a biological control sample,thereby characterizing the disease or disorder. The step of detectingmay comprise performing sequencing of all or some of theoligonucleotides in the complexes, amplification of all or some of theoligonucleotides in the complexes, and/or hybridization of all or someof the oligonucleotides in the complexes to an array. The sequencing maybe high-throughput or next generation sequencing. In some embodiments,the step of detecting comprises visualizing the oligonucleotide orplurality of oligonucleotides in association with the biological testsample. For example, polyligand histochemistry (PHC) as provided by theinvention may be used.

In the methods of the invention, the biological test sample andbiological control sample may each comprise a tissue sample, a cellculture, or a biological fluid. In some embodiments, the biologicalfluid comprises a bodily fluid. Useful bodily fluids within the methodof the invention comprise peripheral blood, sera, plasma, ascites,urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovialfluid, aqueous humor, amniotic fluid, cerumen, breast milk,broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid orpre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair,tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid,lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum,vomit, vaginal secretions, mucosal secretion, stool water, pancreaticjuice, lavage fluids from sinus cavities, bronchopulmonary aspirates,blastocyl cavity fluid, or umbilical cord blood. In some preferredembodiments, the bodily fluid comprises blood, serum or plasma. Thebiological fluid may comprise microvesicles. In such case, the complexesmay be formed between the oligonucleotide or plurality ofoligonucleotides and at least one of the microvesicles.

The biological test sample and biological control sample may furthercomprise isolated microvesicles, wherein optionally the microvesiclesare isolated using at least one of chromatography, filtration,ultrafiltration, centrifugation, ultracentrifugation, flow cytometry,affinity capture (e.g., to a planar surface, column or bead), polymerprecipitation, and using microfluidics. The vesicles can also beisolated after contact with the oligonucleotide or plurality ofoligonucleotides.

The biological test sample and biological control sample may comprisetissue. The tissue can be formalin fixed paraffin embedded (FFPE)tissue. In some embodiments, the FFPE tissue comprises at least one of afixed tissue, unstained slide, bone marrow core or clot, biopsy sample,surgical sample, core needle biopsy, malignant fluid, and fine needleaspirate (FNA). The FFPE tissue can be fixed on a substrate, e.g., aglass slide or membrane.

In various embodiments of the methods of the invention, theoligonucleotide or plurality of oligonucleotides binds a polypeptide orfragment thereof. The polypeptide or fragment thereof can be soluble ormembrane bound, wherein optionally the membrane comprises a cellular ormicrovesicle membrane. The membrane could also be from a fragment of acell, organelle or microvesicle. In some embodiments, the polypeptide orfragment thereof comprises a biomarker in Table 3, Table 4 or any one ofTables 10-17. For example, the polypeptide or fragment thereof could bea general vesicle marker such as in Table 3 or a tissue-related ordisease-related marker such as in Table 4, or a vesicle associatedbiomarker provided in any one of Tables 10-17. The oligonucleotide orplurality of oligonucleotides may bind a microvesicle surface antigen inthe biological sample. For example, the oligonucleotide or plurality ofoligonucleotides can be enriched from a naïve library againstmicrovesicles.

As noted above, the microvesicles may be isolated in whole or in partusing polymer precipitation. In an embodiment, the polymer comprisespolyethylene glycol (PEG). Any appropriate form of PEG may be used. Forexample, the PEG may be PEG 8000. The PEG may be used at any appropriateconcentration. For example, the PEG can be used at a concentration of1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% toisolate the microvesicles. In some embodiments, the PEG is used at aconcentration of 6%.

The disease or disorder detected by the oligonucleotide, plurality ofoligonucleotides, or methods provided here may comprise any appropriatedisease or disorder of interest, including without limitation BreastCancer, Alzheimer's disease, bronchial asthma, Transitional cellcarcinoma of the bladder, Giant cellular osteoblastoclastoma, BrainTumor, Colorectal adenocarcinoma, Chronic obstructive pulmonary disease(COPD), Squamous cell carcinoma of the cervix, acute myocardialinfarction (AMI)/acute heart failure, Chron's Disease, diabetes mellitustype II, Esophageal carcinoma, Squamous cell carcinoma of the larynx,Acute and chronic leukemia of the bone marrow, Lung carcinoma, Malignantlymphoma, Multiple Sclerosis, Ovarian carcinoma, Parkinson disease,Prostate adenocarcinoma, psoriasis, Rheumatoid Arthritis, Renal cellcarcinoma, Squamous cell carcinoma of skin, Adenocarcinoma of thestomach, carcinoma of the thyroid gland, Testicular cancer, ulcerativecolitis, or Uterine adenocarcinoma.

In some embodiments, the disease or disorder comprises a cancer, apremalignant condition, an inflammatory disease, an immune disease, anautoimmune disease or disorder, a cardiovascular disease or disorder,neurological disease or disorder, infectious disease or pain. The cancercan include without limitation one of acute lymphoblastic leukemia;acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancers;AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas;atypical teratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer;brain stem glioma; brain tumor (including brain stem glioma, centralnervous system atypical teratoid/rhabdoid tumor, central nervous systemembryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma,ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymaltumors of intermediate differentiation, supratentorial primitiveneuroectodermal tumors and pineoblastoma); breast cancer; bronchialtumors; Burkitt lymphoma; cancer of unknown primary site; carcinoidtumor; carcinoma of unknown primary site; central nervous systematypical teratoid/rhabdoid tumor; central nervous system embryonaltumors; cervical cancer; childhood cancers; chordoma; chroniclymphocytic leukemia; chronic myelogenous leukemia; chronicmyeloproliferative disorders; colon cancer; colorectal cancer;craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas isletcell tumors; endometrial cancer; ependymoblastoma; ependymoma;esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranialgerm cell tumor; extragonadal germ cell tumor; extrahepatic bile ductcancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinalcarcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinalstromal tumor (GIST); gestational trophoblastic tumor; glioma; hairycell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma;hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposisarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer;lip cancer; liver cancer; lung cancer; malignant fibrous histiocytomabone cancer; medulloblastoma; medulloepithelioma; melanoma; Merkel cellcarcinoma; Merkel cell skin carcinoma; mesothelioma; metastatic squamousneck cancer with occult primary; mouth cancer; multiple endocrineneoplasia syndromes; multiple myeloma; multiple myeloma/plasma cellneoplasm; mycosis fungoides; myelodysplastic syndromes;myeloproliferative neoplasms; nasal cavity cancer; nasopharyngealcancer; neuroblastoma; Non-Hodgkin lymphoma; nonmelanoma skin cancer;non-small cell lung cancer; oral cancer; oral cavity cancer;oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors;ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor;ovarian low malignant potential tumor; pancreatic cancer;papillomatosis; paranasal sinus cancer; parathyroid cancer; pelviccancer; penile cancer; pharyngeal cancer; pineal parenchymal tumors ofintermediate differentiation; pineoblastoma; pituitary tumor; plasmacell neoplasm/multiple myeloma; pleuropulmonary blastoma; primarycentral nervous system (CNS) lymphoma; primary hepatocellular livercancer; prostate cancer; rectal cancer; renal cancer; renal cell(kidney) cancer; renal cell cancer; respiratory tract cancer;retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sézarysyndrome; small cell lung cancer; small intestine cancer; soft tissuesarcoma; squamous cell carcinoma; squamous neck cancer; stomach(gastric) cancer; supratentorial primitive neuroectodermal tumors;T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma;thymoma; thyroid cancer; transitional cell cancer; transitional cellcancer of the renal pelvis and ureter; trophoblastic tumor; uretercancer; urethral cancer; uterine cancer; uterine sarcoma; vaginalcancer; vulvar cancer; Waldenström macroglobulinemia; or Wilm's tumor.The premalignant condition can include without limitation Barrett'sEsophagus. The autoimmune disease can include without limitation one ofinflammatory bowel disease (IBD), Crohn's disease (CD), ulcerativecolitis (UC), pelvic inflammation, vasculitis, psoriasis, diabetes,autoimmune hepatitis, multiple sclerosis, myasthenia gravis, Type Idiabetes, rheumatoid arthritis, psoriasis, systemic lupus erythematosis(SLE), Hashimoto's Thyroiditis, Grave's disease, Ankylosing SpondylitisSjogrens Disease, CREST syndrome, Scleroderma, Rheumatic Disease, organrejection, Primary Sclerosing Cholangitis, or sepsis. The cardiovasculardisease can include without limitation one of atherosclerosis,congestive heart failure, vulnerable plaque, stroke, ischemia, highblood pressure, stenosis, vessel occlusion or a thrombotic event. Theneurological disease can include without limitation one of MultipleSclerosis (MS), Parkinson's Disease (PD), Alzheimer's Disease (AD),schizophrenia, bipolar disorder, depression, autism, Prion Disease,Pick's disease, dementia, Huntington disease (HD), Down's syndrome,cerebrovascular disease, Rasmussen's encephalitis, viral meningitis,neurospsychiatric systemic lupus erythematosus (NPSLE), amyotrophiclateral sclerosis, Creutzfeldt-Jacob disease,Gerstmann-Straussler-Scheinker disease, transmissible spongiformencephalopathy, ischemic reperfusion damage (e.g. stroke), brain trauma,microbial infection, or chronic fatigue syndrome. The pain can includewithout limitation one of fibromyalgia, chronic neuropathic pain, orperipheral neuropathic pain. The infectious disease can include withoutlimitation one of a bacterial infection, viral infection, yeastinfection, Whipple's Disease, Prion Disease, cirrhosis,methicillin-resistant Staphylococcus aureus, HIV, HCV, hepatitis,syphilis, meningitis, malaria, tuberculosis, or influenza. One of skillwill appreciate that the oligonucleotide or plurality ofoligonucleotides or methods of the invention can be used to assess anynumber of these or other related diseases and disorders.

In some embodiments of the invention, the oligonucleotide or pluralityof oligonucleotides and methods of use thereof are useful forcharacterizing certain diseases or disease states. As desired, a pool ofoligonucleotides useful for characterizing various diseases is assembledto create a master pool that can be used to probe useful forcharacterizing the various diseases. One of skill will also appreciatethat pools of oligonucleotides useful for characterizing specificdiseases or disorders can be created as well. The sequences providedherein can also be modified as desired so long as the functional aspectsare still maintained (e.g., binding to various targets or ability tocharacterize a phenotype). For example, the oligonucleotides maycomprise DNA or RNA, incorporate various non-natural nucleotides,incorporate other chemical modifications, or comprise various deletionsor insertions. Such modifications may facilitate synthesis, stability,delivery, labeling, etc, or may have little to no effect in practice. Insome cases, some nucleotides in an oligonucleotide may be substitutedwhile maintaining functional aspects of the oligonucleotide. Similarly,5′ and 3′ flanking regions may be substituted. In still other cases,only a portion of an oligonucleotide may be determined to direct itsfunctionality such that other portions can be deleted or substituted.Numerous techniques to synthesize and modify nucleotides andpolynucleotides are disclosed herein or are known in the art.

In an aspect, the invention provides a kit comprising a reagent forcarrying out the methods of the invention provided herein. In a similaraspect, the invention contemplates use of a reagent for carrying out themethods of the invention provided herein. In embodiments, the reagentcomprises an oligonucleotide or plurality of oligonucleotides. Theoligonucleotide or plurality of oligonucleotides can be those providedherein. The reagent may comprise various other useful componentsincluding without limitation microRNA (miR) and messenger RNA (mRNA)), aprotein-nucleic acid complex, and various combinations, fragments and/orcomplexes of any of these. The one or more reagent can be one or moreof: a) a reagent configured to isolate a microvesicle, optionallywherein the at least one reagent configured to isolate a microvesiclecomprises a binding agent to a microvesicle antigen, a column, asubstrate, a filtration unit, a polymer, polyethylene glycol, PEG4000,PEG8000, a particle or a bead; b) at least one oligonucleotideconfigured to act as a primer or probe in order to amplify, sequence,hybridize or detect the oligonucleotide or plurality ofoligonucleotides; c) a reagent configured to remove one or more abundantprotein from a sample, wherein optionally the one or more abundantprotein comprises at least one of albumin, immunoglobulin, fibrinogenand fibrin; d) a reagent for epitope retrieval; and e) a reagent for PHCvisualization.

Detecting Watson-Crick Base Pairing with an Oligonucleotide Probe

The oligonucleotide probes provided by the invention can bind vianon-Watson Crick base pairing. However, in some cases, theoligonucleotide probes provided by the invention can bind via WatsonCrick base pairing. The oligonucleotide probe libraries of theinvention, e.g., as described above, can query both types of bindingevents simultaneously. For example, some oligonucleotide probes may bindprotein antigens in the classical aptamer sense, whereas otheroligonucleotide probes may bind tissues, cells, microvesicles or othertargets via nucleic acids associated with such targets, e.g., nucleicacid (including without limitation microRNA and mRNA) on the surface ofthe targets. Such surface bound nucleic acids can be associated withproteins. For example, they may comprise Argonaute-microRNA complexes.The argonaute protein can be Ago1, Ago2, Ago3 and/or Ago4.

In addition to the oligonucleotide probe library approach describedherein which relies on determining a sequence of the oligonucleotides(e.g., via sequencing, hybridization or amplification), assays can alsobe designed to detect Watson Crick base pairing. In some embodiments,these approaches rely on Ago2-mediated cleavage wherein an Ago2-microRNAcomplex can be used to detected using oligonucleotide probes. Forfurther details, see PCT/US15/62184, filed Nov. 23, 2015, whichapplication is incorporated by reference herein in its entirety.

Tissue ADAPT

As noted herein, the invention provides methods of enrichingoligonucleotide libraries against various biological samples, includingtissue samples. Tissue samples may be fixed. Fixation may be used in thepreparation of histological sections by which biological tissues arepreserved from decay, thereby preventing autolysis or putrefaction. Theprincipal macromolecules inside a cell are proteins and nucleic acids.Fixation terminates any ongoing biochemical reactions, and may alsoincrease the mechanical strength or stability of the treated tissues.Thus, tissue fixation can be used to preserve cells and tissuecomponents and to do this in such a way as to allow for the preparationof thin, stained sections. Such samples are available for manybiological specimens, e.g., tumor samples. Thus, fixed tissues provide adesirable sample source for various applications of the oligonucleotideprobe libraries of the invention. This process may be referred to as“tissue ADAPT.”

Tissue ADAPT according to the invention has been used to provide variousoligonucleotide probes. In an aspect, the invention provides anoligonucleotide comprising a region corresponding to: a) a variablesequence as described in any one of Examples 19-31; b) a variablesequence as described in any one of Tables 20-23, 25, 27, 38-40, or 45;or c) a sequence listed in any one of SEQ ID NO. 1-206506. In someembodiments, the oligonucleotide further comprises a 5′ region withsequence 5′-CTAGCATGACTGCAGTACGT (SEQ ID NO. 4), a 3′ region withsequence 5′-CTGTCTCTTATACACATCTGACGCTGCCGACGA (SEQ ID NO. 5), or both.The invention further provides an oligonucleotide comprising a nucleicacid sequence or a portion thereof that is at least 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 96, 97, 98, 99 or 100 percent homologous to sucholigonucleotide sequences. In a related aspect, the invention provides aplurality of oligonucleotides comprising at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, orat least 100000 different oligonucleotide sequences as described above.

As described herein, many useful modifications can be made to nucleicacid molecules. In an embodiment, the oligonucleotide or the pluralityof oligonucleotides of the invention comprise a DNA, RNA, 2′-O-methyl orphosphorothioate backbone, or any combination thereof. In someembodiments, the oligonucleotide or the plurality of oligonucleotidescomprises at least one of DNA, RNA, PNA, LNA, UNA, and any combinationthereof. The oligonucleotide or at least one member of the plurality ofoligonucleotides can have at least one functional modification selectedfrom the group consisting of DNA, RNA, biotinylation, a non-naturallyoccurring nucleotide, a deletion, an insertion, an addition, and achemical modification. In some embodiments, the chemical modificationcomprises at least one of C18, polyethylene glycol (PEG), PEG4, PEG6,PEG8, PEG12 and digoxygenin.

The oligonucleotide or plurality of oligonucleotides of the inventioncan be labeled using any useful label such as described herein. Forexample, the oligonucleotide or plurality of oligonucleotides can beattached to a nanoparticle, liposome, gold, magnetic label, fluorescentlabel, light emitting particle, biotin moiety, or radioactive label.

Tissue ADAPT provides for the enrichment of oligonucleotide librariesagainst samples of interest. In an aspect, the invention provides amethod of enriching an oligonucleotide library using multiple rounds ofpositive and negative selection. The method of enriching a plurality ofoligonucleotides may comprise: a) performing at least one round ofpositive selection, wherein the positive selection comprises: i)contacting at least one sample with the plurality of oligonucleotides,wherein the at least one sample comprises tissue; and ii) recoveringmembers of the plurality of oligonucleotides that associated with the atleast one sample; b) optionally performing at least one round ofnegative selection, wherein the negative selection comprises: i)contacting at least one additional sample with the plurality ofoligonucleotides, wherein at least one additional sample comprisestissue; ii) recovering members of the plurality of oligonucleotides thatdid not associate with the at least one additional sample; and c)amplifying the members of the plurality of oligonucleotides recovered inat least one or step (a)(ii) and step (b)(ii), thereby enriching theoligonucleotide library. Various alternatives of these processes areuseful and described herein, such as varying the rounds of enrichment,and varying performance or positive and negative selection steps. In anembodiments, the recovered members of the plurality of oligonucleotidesin step (a)(ii) are used as the input for the next iteration of step(a)(i). In an embodiment, the recovered members of the plurality ofoligonucleotides in step (b)(ii) are used as the input for the nextiteration of step (a)(i). The unenriched oligonucleotide library maypossess great diversity. For example, the unenriched oligonucleotidelibrary may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000,400000, 500000, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵,10¹⁶, 10¹⁷, or at least 10¹⁸ different oligonucleotide sequences. In anembodiment, the unenriched oligonucleotide library comprises the naïveF-Trin library as described herein.

In embodiments of the enrichment methods of the invention, the at leastone sample and/or at least one additional sample comprise tissue. Asdesired, such tissue may be fixed using methods described herein orknown in the art. The fixed tissue may be archived. The fixed tissue maycomprise formalin fixed paraffin embedded (FFPE) tissue. In embodiment,the FFPE tissue comprises at least one of a fixed tissue, unstainedslide, bone marrow core or clot, biopsy sample, surgical sample, coreneedle biopsy, malignant fluid, and fine needle aspirate (FNA). The FFPEtissue can be fixed on a substrate. For example, the substrate can be aglass slide, membrane, or any other useful material.

In some embodiment, the at least one sample and/or the at least oneadditional sample are fixed on different substrates. As a non-limitingexample, the at least one sample is fixed on one glass slide whereas theat least one additional sample is fixed on a different glass slide. Asdesired, such slides may be from different patients, different tumors, asame tumor at different time points, multiple slices of the same tumor,etc. Alternately, the at least one sample and/or the at least oneadditional sample is fixed on a single substrate. As a non-limitingexample, the at least one sample and at least one additional sample arefixed on a same glass slide, such as a tumor sample and normal adjacenttissue to the tumor. In some embodiments, the at least one sample and/orthe at least one additional sample are lysed (see, e.g., Example 29),scraped from a substrate (see, e.g., Example 30), or subjected tomicrodissection (see, e.g., Example 31). Lysed samples can be arrayed ona substrate. The invention contemplates any useful substrate. In someembodiments, the substrate comprises a membrane. For example, themembrane can be a nitrocellulose membrane.

In various embodiments of the enrichment methods of the invention, theat least one sample and the at least one additional sample differ in aphenotype of interest. The at least one sample and the at least oneadditional sample can be from different sections of a same substrate. Asa non-limiting example, the samples may comprise cancer tissue andnormal adjacent tissue from a fixed tissue sample. In such cases, the atleast one sample and the at least one additional sample may be scrapedor microdissected from the same substrate to facilitate enrichment.

The oligonucleotide library can be enriched for analysis of any desiredphenotype. In embodiments, the phenotype comprises a tissue, anatomicalorigin, medical condition, disease, disorder, or any combinationthereof. For example, the tissue can be muscle, epithelial, connectiveand nervous tissue, or any combination thereof. For example, theanatomical origin can be the stomach, liver, small intestine, largeintestine, rectum, anus, lungs, nose, bronchi, kidneys, urinary bladder,urethra, pituitary gland, pineal gland, adrenal gland, thyroid,pancreas, parathyroid, prostate, heart, blood vessels, lymph node, bonemarrow, thymus, spleen, skin, tongue, nose, eyes, ears, teeth, uterus,vagina, testis, penis, ovaries, breast, mammary glands, brain, spinalcord, nerve, bone, ligament, tendon, or any combination thereof. Asdescribed further below, the phenotype can be related to at least one ofdiagnosis, prognosis, theranosis, medical condition, disease ordisorder.

In various embodiments of the enrichment methods of the invention, themethod further comprises determining a target of the enriched members ofthe oligonucleotide library. Techniques for such determining areprovided herein. See, e.g., Examples 9-10, 17 and 19.

Tissue ADAPT further comprises analysis of biological samples. In anaspect, the invention provides a method of characterizing a phenotype ina sample comprising: a) contacting the sample with at least oneoligonucleotide or plurality of oligonucleotides; and b) identifying apresence or level of a complex formed between the at least oneoligonucleotide or plurality of oligonucleotides and the sample, whereinthe presence or level is used to characterize the phenotype. In arelated aspect, the invention provides a method of visualizing a samplecomprising: a) contacting the sample with at least one oligonucleotideor plurality of oligonucleotides; b) removing the at least oneoligonucleotide or members of the plurality of oligonucleotides that donot bind the sample; and c) visualizing the at least one oligonucleotideor plurality of oligonucleotides that bound to the sample. Thevisualization can be used to characterize a phenotype.

The sample to be characterized can be any useful sample, includingwithout limitation a tissue sample, bodily fluid, cell, cell culture,microvesicle, or any combination thereof. In some embodiments, thetissue sample comprises fixed tissue. The tissue may be fixed using anyuseful technique for fixation known in the art. Examples of fixationmethods include heat fixation, immersion, perfusion, chemical fixation,cross-linked (for example, with an aldehyde such as formaldehyde orglutaraldehyde), precipitation (e.g., using an alcohol such as methanol,ethanol and acetone, and acetic acid), oxidation (e.g., using osmiumtetroxide, potassium dichromate, chromic acid, and potassiumpermanganate), mercurials, picrates, Bouin solution, hepes-glutamic acidbuffer-mediated organic solvent protection effect (HOPE), and freezing.In preferred embodiments, the fixed tissue is formalin fixed paraffinembedded (FFPE) tissue. In various embodiments, the FFPE samplecomprises at least one of a fixed tissue, unstained slide, bone marrowcore or clot, biopsy sample, surgical sample, core needle biopsy,malignant fluid, and fine needle aspirate (FNA).

Any useful technique for identifying a presence or level can be used forapplications of tissue ADAPT, including without limitation nucleic acidsequencing, amplification, hybridization, gel electrophoresis,chromatography, or visualization. In some embodiments, the hybridizationcomprises contacting the sample with at least one labeled probe that isconfigured to hybridize with at least one oligonucleotide or pluralityof oligonucleotides. The at least one labeled probe can be directly orindirectly attached to a label. The label can be, e.g., a fluorescent,radioactive or magnetic label. An indirect label can be, e.g., biotin ordigoxigenin. See, e.g., Example 28. In some embodiments, the sequencingcomprises next generation sequencing, dye termination sequencing, and/orpyrosequencing of the at least one oligonucleotide or plurality ofoligonucleotides. The visualization may be that of a signal linkeddirectly or indirectly to the at least one oligonucleotide or pluralityof oligonucleotides. The signal can be any useful signal, e.g., afluorescent signal or an enzymatic signal. In some embodiments, theenzymatic signal is produced by at least one of a luciferase, fireflyluciferase, bacterial luciferase, luciferin, malate dehydrogenase,urease, peroxidase, horseradish peroxidase (HRP), alkaline phosphatase(AP), β-galactosidase, glucoamylase, lysozyme, a saccharide oxidase,glucose oxidase, galactose oxidase, glucose-6-phosphate dehydrogenase, aheterocyclic oxidase, uricase, xanthine oxidase, lactoperoxidase, andmicroperoxidase. Visualization may comprise use of light microscopy orfluorescent microscopy. Various examples of visualization usingpolyligand histochemistry (PHC) are provided herein. See Examples 19-31.

In the methods of the invention directed to characterizing orvisualizing a sample, the target of at least one of the at least oneoligonucleotide or plurality of oligonucleotides may be known. Forexample, an oligonucleotide may bind a known protein target. In someembodiments, the target of at least one the at least one oligonucleotideor plurality of oligonucleotides is unknown. For example, the at leastone oligonucleotide or plurality of oligonucleotides may themselvesprovide a biosignature or other useful result that does not necessarilyrequire knowledge of the antigens bound by some or all of theoligonucleotides. In some embodiments, the targets of a portion of theoligonucleotides are known whereas the targets of another portion of theoligonucleotides have not been identified.

In the methods of characterizing or visualizing a sample, the at leastone oligonucleotide or plurality of oligonucleotides can be as providedherein. The at least one oligonucleotide or plurality ofoligonucleotides may have been determined using the enrichment methodsof the invention provided herein, e.g., enrichment via tissue ADAPT. Forexample, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids may have a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least one of SEQ ID NOs. 1-206506.

For example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids may have a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or allof SEQ ID NOs. 2922-2926, 2929-2947 and 2950-2952. In such cases, thephenotype may be, e.g., lung cancer or prostate cancer. See Example 14.

In another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or allof SEQ ID NOs. 2953-2961 and 2971-2979. In such cases, the phenotype maybe, e.g., prostate cancer. See Example 17.

In yet another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50 or all of SEQ ID NOs. 3039-3061. In such cases, the phenotypemay be, e.g., HER2 status (+/−). See Example 19.

In still another example, the at least one oligonucleotide or pluralityof oligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 150,000 or allof SEQ ID NOs. 3062-103061 and 103062-203061. In such cases, thephenotype may be, e.g., response to anti-HER2 therapy, whereinoptionally the anti-HER2 therapy comprises trastuzumab. See Examples20-22.

In an example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000 or all of SEQ ID NOs. 203064-203067 and203076-206478. In such cases, the phenotype may be, e.g., response to atleast one of FOLFOX and bevacizumab. See Example 24.

In another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or all ofSEQ ID NOs. 206491-206506. In such cases, the phenotype may be, e.g., atissue identity, including without limitation whether the tissuecomprises breast, colon, kidney, lung or pancreatic tissue. See Example29.

In the methods of the invention, including enriching an oligonucleotidelibrary, characterizing a sample or visualizing a sample, the phenotypecan be a biomarker status. In some embodiments, the biomarker isselected from Table 4 or FIGS. 26A-B. In some embodiments, the biomarkerstatus comprises at least one of HER2 positive, HER2 negative, EGFRpositive, EGFR negative, TUBB3 positive, or TUBB3 negative. See, e.g.,Examples 19-23, 24, 26. In some embodiments, the biomarker statuscomprises expression, copy number, mutation, insertion, deletion orother alteration of at least one of ALK, AR, ER, ERCC1, Her2/Neu, MGMT,MLH1, MSH2, MSH6, PD-1, PD-L1, PD-L1 (22c3), PMS2, PR, PTEN, RRM1, TLE3,TOP2A, TOPO1, TrkA, TrkB, TrkC, TS, and TUBB3. In various embodiments,the biomarker status comprises the presence or absence of at least oneof EGFR vIII or MET Exon 14 Skipping. In embodiments, the biomarkerstatus comprises expression, copy number, fusion, mutation, insertion,deletion or other alteration of at least one of ALK, BRAF, NTRK1, NTRK2,NTRK3, RET, ROS1, and RSPO3. In embodiments, the biomarker statuscomprises expression, copy number, fusion, mutation, insertion, deletionor other alteration of at least one of ABL2, ACSL3, ACSL6, AFF1, AFF3,AFF4, AKAP9, AKT2, AKT3, ALDH2, ALK, APC, ARFRP1, ARHGAP26, ARHGEF12,ARID1A, ARID2, ARNT, ASPSCR1, ASXL1, ATF1, ATIC, ATM, ATP1A1, ATR,AURKA, AURKB, AXIN1, AXL, BAP1, BARD1, BCL10, BCL11A, BCL2L11, BCL3,BCL6, BCL7A, BCL9, BCR, BIRC3, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRIP1,BUB1B, C11orf30 (EMSY), C2orf44, CACNA1D, CALR, CAMTA1, CANT1, CARD11,CARS, CASC5, CASP8, CBFA2T3, CBFB, CBL, CBLB, CCDC6, CCNB1IP1, CCND1,CCND2, CCND3, CCNE1, CD274 (PDL1), CD74, CD79A, CDC73, CDH11, CDK4,CDK6, CDK8, CDKN1B, CDKN2A, CDX2, CHEK1, CHEK2, CHIC2, CHN1, CIC, CIITA,CLP1, CLTC, CLTCL1, CNBP, CNTRL, COPB1, CREB1, CREB3L1, CREB3L2, CREBBP,CRKL, CRTC1, CRTC3, CSF1R, CSF3R, CTCF, CTLA4, CTNNA1, CTNNB1, CYLD,CYP2D6, DAXX, DDR2, DDX10, DDX5, DDX6, DEK, DICER1, DOT1L, EBF1, ECT2L,EGFR, ELK4, ELL, EML4, EP300, EPHA3, EPHA5, EPHB1, EPS15, ERBB2 (HER2),ERBB3 (HER3), ERBB4 (HER4), ERC1, ERCC2, ERCC3, ERCC4, ERCC5, ERG, ESR1,ETV1, ETV5, ETV6, EWSR1, EXT1, EXT2, EZH2, EZR, FANCA, FANCC, FANCD2,FANCE, FANCG, FANCL, FAS, FBXO11, FBXW7, FCRL4, FGF10, FGF14, FGF19,FGF23, FGF3, FGF4, FGF6, FGFR1, FGFR1OP, FGFR2, FGFR3, FGFR4, FH, FHIT,FIP1L1, FLCN, FLI1, FLT1, FLT3, FLT4, FNBP1, FOXA1, FOXO1, FOXP1, FUBP1,FUS, GAS7, GATA3, GID4 (C17orf39), GMPS, GNA13, GNAQ, GNAS, GOLGA5,GOPC, GPHN, GPR124, GRIN2A, GSK3B, H3F3A, H3F3B, HERPUD1, HGF, HIP1,HMGA1, HMGA2, HNRNPA2B1, HOOK3, HSP90AA1, HSP90AB1, IDH1, IDH2, IGF1R,IKZF1, IL2, IL21R, IL6ST, IL7R, IRF4, ITK, JAK1, JAK2, JAK3, JAZF1,KDM5A, KDR (VEGFR2), KEAP1, KIAA1549, KIF5B, KIT, KLHL6, KMT2A (MLL),KMT2C (MLL3), KMT2D (MLL2), KRAS, KTN1, LCK, LCP1, LGR5, LHFP, LIFR,LPP, LRIG3, LRP1B, LYL1, MAF, MALT1, MAML2, MAP2K1, MAP2K2, MAP2K4,MAP3K1, MCL1, MDM2, MDM4, MDS2, MEF2B, MEN1, MET (cMET), MITF, MLF1,MLH1 (NGS), MLLT1, MLLT10, MLLT3, MLLT4, MLLT6, MNX1, MRE11A, MSH2(NGS), MSH6 (NGS), MSI2, MTOR, MYB, MYC, MYCN, MYD88, MYH11, MYH9, NACA,NCKIPSD, NCOA1, NCOA2, NCOA4, NF1, NF2, NFE2L2, NFIB, NFKB2, NFKBIA,NIN, NOTCH2, NPM1, NR4A3, NSD1, NT5C2, NTRK1, NTRK2, NTRK3, NUP214,NUP93, NUP98, NUTM1, PALB2, PAX3, PAX5, PAX7, PBRM1, PBX1, PCM1, PCSK7,PDCD1 (PD1), PDCD1LG2 (PDL2), PDGFB, PDGFRA, PDGFRB, PDK1, PER1, PICALM,PIK3CA, PIK3R1, PIK3R2, PIM1, PML, PMS2 (NGS), POLE, POT1, POU2AF1,PPARG, PRCC, PRDM1, PRDM16, PRKAR1A, PRRX1, PSIP1, PTCH1, PTEN (NGS),PTPN11, PTPRC, RABEP1, RAC1, RAD50, RAD51, RAD51B, RAF1, RALGDS,RANBP17, RAP1GDS1, RARA, RB1, RBM15, REL, RET, RICTOR, RMI2, RNF43,ROS1, RPL22, RPL5, RPN1, RPTOR, RUNX1, RUNX1T1, SBDS, SDC4, SDHAF2,SDHB, SDHC, SDHD, SEPT9, SET, SETBP1, SETD2, SF3B1, SH2B3, SH3GL1,SLC34A2, SMAD2, SMAD4, SMARCB1, SMARCE1, SMO, SNX29, SOX10, SPECC1,SPEN, SRGAP3, SRSF2, SRSF3, SS18, SS18L1, STAT3, STAT4, STAT5B, STIL,STK11, SUFU, SUZ12, SYK, TAF15, TCF12, TCF3, TCF7L2, TET1, TET2, TFEB,TFG, TFRC, TGFBR2, TLX1, TNFAIP3, TNFRSF14, TNFRSF17, TOP1, TP53, TPM3,TPM4, TPR, TRAF7, TRIM26, TRIM27, TRIM33, TRIP11, TRRAP, TSC1, TSC2,TSHR, TTL, U2AF1, USP6, VEGFA, VEGFB, VTI1A, WHSC1, WHSC1L1, WIF1,WISP3, WRN, WT1, WWTR1, XPA, XPC, XPO1, YWHAE, ZMYM2, ZNF217, ZNF331,ZNF384, ZNF521, and ZNF703. The biomarker status may compriseexpression, copy number, fusion, mutation, insertion, deletion or otheralteration of at least one of ABI1, ABL1, ACKR3, AKT1, AMER1 (FAM123B),AR, ARAF, ATP2B3, ATRX, BCL11B, BCL2, BCL2L2, BCOR, BCORL1, BRD3, BRD4,BTG1, BTK, C15orf65, CBLC, CD79B, CDH1, CDK12, CDKN2B, CDKN2C, CEBPA,CHCHD7, CNOT3, COL1A1, COX6C, CRLF2, DDB2, DDIT3, DNM2, DNMT3A, EIF4A2,ELF4, ELN, ERCC1 (NGS), ETV4, FAM46C, FANCF, FEV, FOXL2, FOXO3, FOXO4,FSTL3, GATA1, GATA2, GNA11, GPC3, HEY1, HIST1H3B, HIST1H4I, HLF,HMGN2P46, HNF1A, HOXA11, HOXA13, HOXA9, HOXC11, HOXC13, HOXD11, HOXD13,HRAS, IKBKE, INHBA, IRS2, JUN, KAT6A (MYST3), KAT6B, KCNJ5, KDM5C,KDM6A, KDSR, KLF4, KLK2, LASP1, LMO1, LMO2, MAFB, MAX, MECOM, MED12,MKL1, MLLT11, MN1, MPL, MSN, MTCP1, MUC1, MUTYH, MYCL (MYCL1), NBN,NDRG1, NKX2-1, NONO, NOTCH1, NRAS, NUMA1, NUTM2B, OLIG2, OMD, P2RY8,PAFAH1B2, PAK3, PATZ1, PAX8, PDE4DIP, PHF6, PHOX2B, PIK3CG, PLAG1, PMS1,POU5F1, PPP2R1A, PRF1, PRKDC, RAD21, RECQL4, RHOH, RNF213, RPL10, SEPT5,SEPT6, SFPQ, SLC45A3, SMARCA4, SOCS1, SOX2, SPOP, SRC, SSX1, STAG2,TAL1, TAL2, TBL1XR1, TCEA1, TCL1A, TERT, TFE3, TFPT, THRAP3, TLX3,TMPRSS2, UBR5, VHL, WAS, ZBTB16, and ZRSR2. The biomarker status can befor a biomarker in any one of PCT/US2007/69286, filed May 18, 2007;PCT/US2009/60630, filed Oct. 14, 2009; PCT/2010/000407, filed Feb. 11,2010; PCT/US12/41393, filed Jun. 7, 2012; PCT/US2013/073184, filed Dec.4, 2013; PCT/US2010/54366, filed Oct. 27, 2010; PCT/US11/67527, filedDec. 28, 2011; PCT/US15/13618, filed Jan. 29, 2015; and PCT/US16/20657,filed Mar. 3, 2016; each of which applications is incorporated herein byreference in its entirety. Examples of additional biomarkers that can beincorporated into the methods and compositions of the invention includewithout limitation those disclosed in International Patent ApplicationNos. PCT/US2009/62880, filed Oct. 30, 2009; PCT/US2009/006095, filedNov. 12, 2009; PCT/US2011/26750, filed Mar. 1, 2011; PCT/US2011/031479,filed Apr. 6, 2011; PCT/US11/48327, filed Aug. 18, 2011;PCT/US2008/71235, filed Jul. 25, 2008; PCT/US10/58461, filed Nov. 30,2010; PCT/US2011/21160, filed Jan. 13, 2011; PCT/US2013/030302, filedMar. 11, 2013; PCT/US12/25741, filed Feb. 17, 2012; PCT/2008/76109,filed Sep. 12, 2008; PCT/US12/42519, filed Jun. 14, 2012;PCT/US12/50030, filed Aug. 8, 2012; PCT/US12/49615, filed Aug. 3, 2012;PCT/US12/41387, filed Jun. 7, 2012; PCT/US2013/072019, filed Nov. 26,2013; PCT/US2014/039858, filed May 28, 2013; PCT/IB2013/003092, filedOct. 23, 2013; PCT/US13/76611, filed Dec. 19, 2013; PCT/US14/53306,filed Aug. 28, 2014; and PCT/US15/62184, filed Nov. 23, 2015;PCT/US16/40157, filed Jun. 29, 2016; PCT/US16/44595, filed Jul. 28,2016; and PCT/US16/21632, filed Mar. 9, 2016; each of which applicationsis incorporated herein by reference in its entirety. The methods of theinvention can be used to enrich oligonucleotide libraries and analyzesamples given any desired biomarker status for which appropriate samplesare available.

In the methods of the invention, including enriching an oligonucleotidelibrary, characterizing a sample or visualizing a sample, the phenotypecan be a phenotype comprises a disease or disorder. The methods can beemployed to assist in providing a diagnosis, prognosis and/or theranosisfor the disease or disorder. For example, the enriching may be performedusing sample such that the enriched library can be used to assist inproviding a diagnosis, prognosis and/or theranosis for the disease ordisorder. Similarly, the characterizing may comprise assisting inproviding a diagnosis, prognosis and/or theranosis for the disease ordisorder. The visualization may also comprise assisting in providing adiagnosis, prognosis and/or theranosis for the disease or disorder. Insome embodiments, the theranosis comprises predicting a treatmentefficacy or lack thereof, classifying a patient as a responder ornon-responder to treatment, or monitoring a treatment efficacy. Thetheranosis can be directed to any appropriate treatment, e.g., thetreatment may comprise at least one of chemotherapy, immunotherapy,targeted cancer therapy, a monoclonal antibody, an anti-HER2 antibody,trastuzumab, an anti-VEGF antibody, bevacizumab, and/or platinum/taxanetherapy. In some embodiments, the treatment comprises at least one ofafatinib, afatinib+cetuximab, alectinib, aspirin, atezolizumab,bicalutamide, cabozantinib, capecitabine, carboplatin, ceritinib,cetuximab, cisplatin, crizotinib, dabrafenib, dacarbazine, doxorubicin,enzalutamide, epirubicin, erlotinib, everolimus, exemestane+everolimus,fluorouracil, fulvestrant, gefitinib, gemcitabine, hormone therapies,irinotecan, lapatinib, liposomal-doxorubicin, matinib, mitomycin-c,nab-paclitaxel, nivolumab, olaparib, osimertinib, oxaliplatin,palbociclib combination therapy, paclitaxel, palbociclib, panitumumab,pembrolizumab, pemetrexed, pertuzumab, sunitinib, T-DM1, temozolomidedocetaxel, temsirolimus, topotecan, trametinib, trastuzumab, vandetanib,and vemurafenib. The hormone therapy can be one or more of tamoxifen,toremifene, fulvestrant, letrozole, anastrozole, exemestane, megestrolacetate, leuprolide, goserelin, bicalutamide, flutamide, abiraterone,enzalutamide, triptorelin, abarelix, and degarelix.

The theranosis can be for a therapy listed in FIGS. 26A-B, or in any oneof PCT/US2007/69286, filed May 18, 2007; PCT/US2009/60630, filed Oct.14, 2009; PCT/2010/000407, filed Feb. 11, 2010; PCT/US12/41393, filedJun. 7, 2012; PCT/US2013/073184, filed Dec. 4, 2013; PCT/US2010/54366,filed Oct. 27, 2010; PCT/US11/67527, filed Dec. 28, 2011;PCT/US15/13618, filed Jan. 29, 2015; and PCT/US16/20657, filed Mar. 3,2016; each of which applications is incorporated herein by reference inits entirety. The likelihood of benefit or lack of benefit of thesetherapies for treating various cancers can be related to a biomarkerstatus. For example, anti-HER2 treatments may be of most benefit forpatients whose tumors express HER2, and vice versa. Using appropriatesamples for enrichment (e.g., known responders or non-responders),tissue ADAPT may be used to provide improved theranosis as compared toconventional companion diagnostics. See, e.g., Examples 20-22; see alsoExamples 24, 27.

In the methods of the invention directed to characterizing a sample, thecharacterizing may comprise comparing the presence or level to areference. In some embodiments, the reference comprises a presence orlevel determined in a sample from an individual without a disease ordisorder, or from an individual with a different state of a disease ordisorder. The presence or level can be that of a visual level, such asan IHC score, determined by the visualizing. As a non-limiting example,the comparison to the reference of at least one oligonucleotide orplurality of oligonucleotides provided by the invention indicates thatthe sample comprises a cancer sample or a non-cancer/normal sample.

In some embodiments of the methods of the invention, one or more samplecomprises a bodily fluid. The bodily fluid can be any useful bodilyfluid, including without limitation peripheral blood, sera, plasma,ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow,synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk,broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid orpre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair oil,tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid,lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum,vomit, vaginal secretions, mucosal secretion, stool water, pancreaticjuice, lavage fluids from sinus cavities, bronchopulmonary aspirates,blastocyl cavity fluid, or umbilical cord blood.

In the methods of the invention, including characterizing a sample orvisualizing a sample, the sample can be from a subject suspected ofhaving or being predisposed to a medical condition, disease, ordisorder.

In the methods of the invention, including enriching an oligonucleotidelibrary, characterizing a sample or visualizing a sample, the medicalcondition, the disease or disorder may be a cancer, a premalignantcondition, an inflammatory disease, an immune disease, an autoimmunedisease or disorder, a cardiovascular disease or disorder, neurologicaldisease or disorder, infectious disease or pain. In some embodiments,the cancer comprises an acute lymphoblastic leukemia; acute myeloidleukemia; adrenocortical carcinoma; AIDS-related cancers; AIDS-relatedlymphoma; anal cancer; appendix cancer; astrocytomas; atypicalteratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer; brainstem glioma; brain tumor (including brain stem glioma, central nervoussystem atypical teratoid/rhabdoid tumor, central nervous systemembryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma,ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymaltumors of intermediate differentiation, supratentorial primitiveneuroectodermal tumors and pineoblastoma); breast cancer; bronchialtumors; Burkitt lymphoma; cancer of unknown primary site; carcinoidtumor; carcinoma of unknown primary site; central nervous systematypical teratoid/rhabdoid tumor; central nervous system embryonaltumors; cervical cancer; childhood cancers; chordoma; chroniclymphocytic leukemia; chronic myelogenous leukemia; chronicmyeloproliferative disorders; colon cancer; colorectal cancer;craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas isletcell tumors; endometrial cancer; ependymoblastoma; ependymoma;esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranialgerm cell tumor; extragonadal germ cell tumor; extrahepatic bile ductcancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinalcarcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinalstromal tumor (GIST); gestational trophoblastic tumor; glioma; hairycell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma;hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposisarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer;lip cancer; liver cancer; lung cancer; malignant fibrous histiocytomabone cancer; medulloblastoma; medulloepithelioma; melanoma; Merkel cellcarcinoma; Merkel cell skin carcinoma; mesothelioma; metastatic squamousneck cancer with occult primary; mouth cancer; multiple endocrineneoplasia syndromes; multiple myeloma; multiple myeloma/plasma cellneoplasm; mycosis fungoides; myelodysplastic syndromes;myeloproliferative neoplasms; nasal cavity cancer; nasopharyngealcancer; neuroblastoma; Non-Hodgkin lymphoma; nonmelanoma skin cancer;non-small cell lung cancer; oral cancer; oral cavity cancer;oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors;ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor;ovarian low malignant potential tumor; pancreatic cancer;papillomatosis; paranasal sinus cancer; parathyroid cancer; pelviccancer; penile cancer; pharyngeal cancer; pineal parenchymal tumors ofintermediate differentiation; pineoblastoma; pituitary tumor; plasmacell neoplasm/multiple myeloma; pleuropulmonary blastoma; primarycentral nervous system (CNS) lymphoma; primary hepatocellular livercancer; prostate cancer; rectal cancer; renal cancer; renal cell(kidney) cancer; renal cell cancer; respiratory tract cancer;retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sézarysyndrome; small cell lung cancer; small intestine cancer; soft tissuesarcoma; squamous cell carcinoma; squamous neck cancer; stomach(gastric) cancer; supratentorial primitive neuroectodermal tumors;T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma;thymoma; thyroid cancer; transitional cell cancer; transitional cellcancer of the renal pelvis and ureter; trophoblastic tumor; uretercancer; urethral cancer; uterine cancer; uterine sarcoma; vaginalcancer; vulvar cancer; Waldenström macroglobulinemia; or Wilm's tumor.In some embodiments, the premalignant condition comprises Barrett'sEsophagus. In some embodiments, the autoimmune disease comprisesinflammatory bowel disease (IBD), Crohn's disease (CD), ulcerativecolitis (UC), pelvic inflammation, vasculitis, psoriasis, diabetes,autoimmune hepatitis, multiple sclerosis, myasthenia gravis, Type Idiabetes, rheumatoid arthritis, psoriasis, systemic lupus erythematosis(SLE), Hashimoto's Thyroiditis, Grave's disease, Ankylosing SpondylitisSjogrens Disease, CREST syndrome, Scleroderma, Rheumatic Disease, organrejection, Primary Sclerosing Cholangitis, or sepsis. In someembodiments, the cardiovascular disease comprises atherosclerosis,congestive heart failure, vulnerable plaque, stroke, ischemia, highblood pressure, stenosis, vessel occlusion or a thrombotic event. Insome embodiments, the neurological disease comprises Multiple Sclerosis(MS), Parkinson's Disease (PD), Alzheimer's Disease (AD), schizophrenia,bipolar disorder, depression, autism, Prion Disease, Pick's disease,dementia, Huntington disease (HD), Down's syndrome, cerebrovasculardisease, Rasmussen's encephalitis, viral meningitis, neurospsychiatricsystemic lupus erythematosus (NPSLE), amyotrophic lateral sclerosis,Creutzfeldt-Jacob disease, Gerstmann-Straussler-Scheinker disease,transmissible spongiform encephalopathy, ischemic reperfusion damage(e.g. stroke), brain trauma, microbial infection, or chronic fatiguesyndrome. In some embodiments, the pain comprises fibromyalgia, chronicneuropathic pain, or peripheral neuropathic pain. In some embodiments,the infectious disease comprises a bacterial infection, viral infection,yeast infection, Whipple's Disease, Prion Disease, cirrhosis,methicillin-resistant Staphylococcus aureus, HIV, HCV, hepatitis,syphilis, meningitis, malaria, tuberculosis, influenza.

In an aspect, the invention provides a kit comprising at least onereagent for carrying out the methods provided by the invention,including enriching an oligonucleotide library, characterizing a sampleor visualizing a sample. In a related aspect, the invention provides useof at least one reagent for carrying out the methods provided by theinvention, including enriching an oligonucleotide library,characterizing a sample or visualizing a sample. In some embodiments,the at least one reagent comprises an oligonucleotide or a plurality ofoligonucleotides provided herein. Additional useful reagents are alsoprovided herein. See, e.g., the protocols provided in the Examples.

The at least one oligonucleotide or plurality of oligonucleotidesprovided by tissue ADAPT can be used for various purposes. As describedabove, such oligonucleotides can be used to characterize and/orvisualize a sample. As the oligonucleotides are selected to associatewith tissues of interest, such associations can also be used for otherpurposes. In an aspect, the invention provides a method of imaging atleast one cell or tissue, comprising contacting the at least one cell ortissue with at least one oligonucleotide or plurality ofoligonucleotides provided herein, and detecting the at least oneoligonucleotide or the plurality of oligonucleotides in contact with atleast one cell or tissue. In a non-limiting example, such method can beused for medical imaging of a tumor or tissue in a patient.

For example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids may have a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or allof SEQ ID NOs. 2922-2926, 2929-2947 and 2950-2952. In such cases, theimaging may be, e.g., directed to lung or prostate tissue. See Example14.

In another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or allof SEQ ID NOs. 2953-2961 and 2971-2979. In such cases, the phenotype maybe, e.g., prostate cancer. See Example 17.

In yet another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50 or all of SEQ ID NOs. 3039-3061. In such cases, the imagingmay be, e.g., directed to HER2 status of a cell or tissue. See Example19.

In still another example, the at least one oligonucleotide or pluralityof oligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 150,000 or allof SEQ ID NOs. 3062-103061 and 103062-203061. In such cases, the imagingmay be, e.g., directed to a HER2 status of a cell or tissue. SeeExamples 20-22.

In an example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000 or all of SEQ ID NOs. 203064-203067 and203076-206478. In such cases, the imaging may be, e.g., directed tocolorectal cells or tissue. See Example 24.

In another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or all ofSEQ ID NOs. 206491-206506. In such cases, the imaging may be, e.g.,directed to a tissue, including without limitation breast, colon,kidney, lung or pancreatic tissue. See Example 29.

In the imaging methods provided by the invention, the at least oneoligonucleotide or the plurality of oligonucleotides can carry varioususeful chemical structures or modifications such as described herein.Such modifications can be made to enhance binding, stability, allowdetection, or for other useful purposes.

In the imaging methods provided by the invention, the at least oneoligonucleotide or the plurality of oligonucleotides can be administeredto a subject prior to the detecting. Such method may allow imaging of atleast one cell or tissue in the subject. In some embodiments, the atleast one cell or tissue comprises neoplastic, malignant, tumor,hyperplastic, or dysplastic cells. In some embodiments, the at least onecell or tissue comprises at least one of lymphoma, leukemia, renalcarcinoma, sarcoma, hemangiopericytoma, melanoma, abdominal cancer,gastric cancer, colon cancer, cervical cancer, prostate cancer,pancreatic cancer, breast cancer, or non-small cell lung cancer cells.The at least one cell or tissue can be from any desired tissue orrelated to desired any medical condition, disease or disorder such asdescribed herein.

As the oligonucleotides provided by tissue ADAPT are selected toassociate with tissues of interest, such associations can also be usedin therapeutic applications such as targeted drug delivery. Theoligonucleotides may provide therapeutic benefit alone or by providingtargeted delivery of immunomodulators, drugs and the like. In an aspect,the invention provides a pharmaceutical composition comprising atherapeutically effective amount of a construct comprising the at leastone oligonucleotide or the plurality of oligonucleotides as providedherein, or a salt thereof, and a pharmaceutically acceptable carrier,diluent, or both. In some embodiments, the at least one oligonucleotideor plurality of oligonucleotides associates with one or more proteinlisted in Table 28.

For example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or allof SEQ ID NOs. 2922-2926, 2929-2947 and 2950-2952. Such pharmaceuticalcomposition may be useful for therapy related to a cancer, whereinoptionally the cancer comprises lung cancer or prostate cancer. SeeExample 14.

In another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or allof SEQ ID NOs. 2953-2961 and 2971-2979. Such pharmaceutical compositionmay be useful for therapy related to a cancer, wherein optionally thecancer comprises prostate cancer. See Example 17.

In still another example, the at least one oligonucleotide or pluralityof oligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50 or all of SEQ ID NOs. 3039-3061. Such pharmaceuticalcomposition may be useful for therapy related to a cancer, whereinoptionally the cancer comprises breast cancer. See Example 19.

In yet another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 150,000 or allof SEQ ID NOs. 3062-103061 and 103062-203061. Such pharmaceuticalcomposition may be useful for therapy related to a cancer, whereinoptionally the cancer comprises breast cancer. See Examples 20-22.

In an example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000 or all of SEQ ID NOs. 203064-203067 and203076-206478. Such pharmaceutical composition may be useful for therapyrelated to a cancer, wherein optionally the cancer comprises colorectalcancer. See Example 24.

In yet another example, the at least one oligonucleotide or plurality ofoligonucleotides may comprise nucleic acids having a sequence or aportion thereof that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100 percent homologous to an oligonucleotide sequenceaccording to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or all ofSEQ ID NOs. 206491-206506. Such pharmaceutical composition may be usefulfor therapy related to a cancer, wherein optionally the cancer comprisesa cancer of the breast, colon, kidney, lung or pancreas. See Example 29.

The at least one oligonucleotide or the plurality of oligonucleotideswithin the pharmaceutical composition can have any useful desiredchemical modification. In an embodiment, the at least oneoligonucleotide or the plurality of oligonucleotides is attached to atoxin or chemotherapeutic agent. The at least one oligonucleotide or theplurality of oligonucleotides may be comprised within a multipartiteconstruct. The at least one oligonucleotide or the plurality ofoligonucleotides can be attached to a liposome or nanoparticle. In someembodiments, the liposome or nanoparticle comprises a toxin orchemotherapeutic agent. In such cases, the at least one oligonucleotideor the plurality of oligonucleotides can be used to target a therapeuticagent to a desired cell, tissue, organ or the like.

In a related aspect, the invention provides a method of treating orameliorating a disease or disorder in a subject in need thereof,comprising administering the pharmaceutical composition of the inventionto the subject. In another related aspect, the invention provides amethod of inducing cytotoxicity in a subject, comprising administeringthe pharmaceutical composition of the invention to the subject. Anyuseful means of administering can be used, including without limitationat least one of intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, oral, sublingual,intracerebral, intravaginal, transdermal, rectal, by inhalation, topicaladministration, or any combination thereof.

The oligonucleotide or plurality of oligonucleotides provided by tissueADAPT can be used for imaging or therapeutic applications of any desiredmedical condition, disease or disorder, such as those described herein(see above). As a non-limiting example, the oligonucleotide or pluralityof oligonucleotides can be used for imaging of tumors from variousanatomical locals, or for treatment of cancers derived from varioustissues.

Kits

The invention also provides a kit comprising one or more reagent tocarry out the methods of the invention. For example, the one or morereagent can be the one or more aptamer, a buffer, blocker, enzyme, orcombination thereof. The one or more reagent may comprise any usefulreagents for carrying out the subject methods, including withoutlimitation aptamer libraries, substrates such as microbeads or planararrays or wells, reagents for biomarker and/or microvesicle isolation(e.g., via chromatography, filtration, ultrafiltration, centrifugation,ultracentrifugation, flow cytometry, affinity capture (e.g., to a planarsurface, column or bead), polymer precipitation, and/or usingmicrofluidics), aptamers directed to specific targets, aptamer poolsthat facilitate detection of a tissue/cell/microvesicle/biomarkerpopulation, reagents such as primers for nucleic acid sequencing oramplification, arrays for nucleic acid hybridization, detectable labels,solvents or buffers and the like, various linkers, various assaycomponents, blockers, and the like. The one or more reagent may alsocomprise various compositions provided by the invention. In anembodiment, the one or more reagent comprises one or more aptamer of theinvention. The one or more reagent can comprise a substrate, such as aplanar substrate, column or bead. The kit can contain instructions tocarry out various assays using the one or more reagent. The one or morereagent may comprise a reagent for performing a PHC assay, includingcomponents of enzymatic detection systems and substrates thereof usefulfor staining a tissue sample.

In an embodiment, the kit comprises an oligonucleotide probe orcomposition provided herein. The kit can be configured to carry out themethods provided herein. For example, the kit can include an aptamer ofthe invention, a substrate, or both an aptamer of the invention and asubstrate.

In an embodiment, the kit is configured to carry out an assay. Forexample, the kit can contain one or more reagent and instructions fordetecting the presence or level of a biological entity in a biologicalsample. In such cases, the kit can include one or more binding agent toa biological entity of interest. The one or more binding agent can bebound to a substrate. The one or more binding agent can be modified toallow capture, detection or visualization. For example, the one or morebinding agent can be biotinylated or conjugated to digoxigenin.

In an embodiment, the kit comprises a set of oligonucleotides thatprovide a particular oligonucleotide profile for a biological sample. Anoligonucleotide profile can include, without limitation, a profile thatcan be used to characterize a particular disease or disorder. Forexample, the disease or disorder can be a proliferative disease ordisorder, including without limitation a cancer. In some embodiments,the cancer comprises a breast cancer.

EXAMPLES Example 1 Aptamer Target Identification

In this Example, aptamers conjugated to microspheres are used to assistin determining the target of two aptamers identified by libraryscreening methods as described above. The general approach is shown inFIG. 9. The approach is used to verify the targets of CAR003, an aptameridentified by library screening to recognize EpCAM. CAR003 is an aptamercandidate identified using the above methodology. As an RNA aptamer,CAR003 with alternate tail sequence has the following RNA sequence (SEQID NO. 3):

5′-auccagagug acgcagcagu cuuuucugau ggacacgugguggucuagua ucacuaagcc accgugucca-3′

In this approach, the sequence of CAR003 is randomly rearranged beforelinkage to the microspheres. The microspheres are used as controls tobind to targets that are similar but not identical to the intendedtarget molecule.

The protocol used is as follows:

1) The candidate aptamers (here, CAR003) and negative control aptamers(here, randomly arranged CAR003) are synthesized with modifications toallow capture (here, the aptamers are biotinylated) and crosslinking(here, using the Sulfo-SBED Biotin Label Transfer Reagent and Kit,Catalog Number 33073 from Thermo Fisher Scientific Inc., Rockford, Ill.,to allow photocrosslinking)

2) Each of the aptamers is individually mixed with microvesicles havingthe target of interest (here, BrCa cell line microvesicles).

3) After incubation to allow the aptamers to bind target, ultravioletlight is applied to the mixtures to trigger crosslinking of the aptamerswith the microvesicle targets.

4) The microvesicles are lysed, thereby releasing the crosslinkedaptamer-target complex into solution.

5) The crosslinked aptamer-target complexes are captured from solutionusing a streptavidin coated substrate.

6) The crosslinked aptamer-target complexes for each aptamer are runindividually on SDS-PAGE gel electrophoresis. The captured proteintargets are visualized with Coomasie Blue staining.

7) The crosslinking and binding steps may be promiscuous so thatmultiple bands including the intended target but also random proteinswill appear on each of the gels. The intended target will be found in aband that appears on the gel with the candidate aptamer (here, CAR003)but not the related negative control aptamers (here, randomly arrangedCAR003). The bands corresponding to the target are excised from the gel.

8) Mass spectrometry (MS) is used to identify the aptamer target fromthe excised bands.

Example 2 Disease Diagnosis

This example illustrates the use of oligonucleotide probes of theinvention to diagnose a proliferative disease.

A suitable quantity of an oligonucleotide or pool of oligonucleotidesthat bind a BrCa-derived population of microvesicles, such as identifiedin Example 12 or various Examples below, is synthesized via chemicalmeans known in the art. The oligonucleotides are conjugated to adiagnostic agent suitable for detection, such as a fluorescent moiety,using a conjugation method known in the art.

The composition is applied to microvesicles isolated from blood samplestaken from a test cohort of patients suffering from a proliferativedisease associated with the overexpression of microvesicles, e.g. breastcancer. The composition is likewise applied to microvesicles isolatedfrom blood samples taken from a negative control cohort, not sufferingfrom a proliferative disease.

The use of appropriate detection techniques (e.g., microbead assay orflow cytometry) on the test cohort samples indicates the presence ofdisease, while the same techniques applied to the control cohort samplesindicate the absence of disease.

The results show that the oligonucleotides of the present invention areuseful in diagnosing proliferative diseases.

Example 3 Theranostics

This example illustrates the use of oligonucleotide probes of thepresent invention to provide a theranosis for a drug for treating aproliferative disease.

A suitable quantity of an oligonucleotide or pool of oligonucleotidesthat bind breast cancer tissue, such as identified in Examples 19-21 orvarious Examples below, is synthesized via chemical means known in theart. The probes are conjugated to an agent suitable for detection, suchas a biotin moiety, which can then be detected using streptavidinconstructs such as streptavidin-horse radish peroxidase usingimmunohistochemistry (IHC) techniques. The oligonucleotide probe orpanel of oligonucleotide probes are within a suitable composition, suchas a buffered solution.

Treatment selection. The probes are applied to tumor tissue samplestaken from a test cohort of patients suffering from a proliferativedisease, e.g. breast cancer, that responded to a certain treatment,e.g., trastuzumab. The probes are likewise applied to tumor tissue takenfrom a control cohort consisting of patients suffering from the sameproliferative disease that did not respond to the treatment. The use ofappropriate detection techniques (e.g., IHC) on the test cohort samplesindicates that probes which bind the samples are useful for identifyingpatients that will respond to the treatment, while the same techniquesapplied to the control cohort samples identifies probes useful foridentifying patients that will not respond to the treatment.

Treatment monitoring. In another setting, the probes are applied totumor tissue samples from a test cohort of patients suffering from aproliferative disease, e.g. breast cancer, prior to or during a courseof treatment, such as surgery, radiotherapy and/or chemotherapy. Theprobes are then applied to tumor tissue samples from the patients over atime course. The use of appropriate detection techniques (e.g., IHC) onthe test cohort samples indicates whether the detected population ofdisease-related cells increases, decreases, or remains steady inconcentration over time during the course of treatment. An increase inthe population of disease-related cells post-treatment may indicate thatthe treatment is less effective whereas a decrease in the population ofdisease-related cells post-treatment may indicate that the treatment hasa beneficial effect.

The results show that the oligonucleotide probes of the presentinvention are useful in theranosing proliferative diseases.

Example 4 Therapeutic Oligonucleotide Probes

This example illustrates the use of oligonucleotide probes of thepresent invention to treat a proliferative disease.

A suitable quantity of an oligonucleotide or pool of oligonucleotidesthat bind breast cancer tumor tissue, such as identified in Examples19-21 or various Examples below, is synthesized via chemical means knownin the art. The oligonucleotides are conjugated to a chemotherapeuticagent, such as Doxil, using a conjugation method known in the art. Theconjugate is formulated in an aqueous composition.

The composition is administered intravenously, in one or more doses, toa test cohort of subjects suffering from breast cancer. A control cohortsuffering from breast cancer is administered a placebo intravenously,according to a corresponding dosage regimen.

Pathological analysis of tumor samples and/or survival indicates thatmortality and/or morbidity are improved in the test cohort over thecontrol cohort.

The results show that the oligonucleotides of the present invention areuseful in treating proliferative diseases.

Example 5 Oligonucleotide—Sequencing Detection Method

This example illustrates the use of an oligonucleotide pool to detectmicrovesicles that are indicative of a phenotype of interest. The methodmakes use of a pool of oligonucleotides that have been enriched againsta target of interest that is indicative of a phenotype of interest. Themethod in this Example allows efficient use of a library ofoligonucleotides to preferentially recognize a target entity.

For purposes of illustration, the method is described in the Examplewith a microvesicle target from a bodily fluid sample. One of skill willappreciate that the method can be extended to other types of targetentity (e.g., cells, proteins, various other biological complexes),sample (e.g., tissue, cell culture, biopsy, other bodily fluids) andother phenotypes (other cancers, other diseases, etc) by enriching anaptamer library against the desired input samples.

General workflow:

1) Obtain sample (plasma, serum, urine or any other biological sample)of patients with unknown medical etymology and pre-treating themaccordingly to ensure availability of the target of interest (seebelow). Where the target of interest is a microvesicle population, themicrovesicles can be isolated and optionally tethered to a solid supportsuch as a microbead.

2) Expose pre-treated sample to an oligonucleotide pool carrying certainspecificity against target of interest. As described herein, anoligonucleotide pool carrying certain specificity against the target ofinterest can be enriched using various selection schemes, e.g., usingnon-cancer microvesicles for negative selection and cancer microvesiclesfor positive selection as described above. DNA or RNA oligonucleotidescan be used as desired.

3) Contact oligonucleotide library with the sample.

4) Elute any oligonucleotides bound to the target.

5) Sequence the eluted oligonucleotides. Next generation sequencingmethods can be used.

6) Analyze oligonucleotide profile from the sequencing. A profile ofoligonucleotides known to bind the target of interest indicates thepresence of the target within the input sample. The profile can be usedto characterize the sample, e.g., as cancer or non-cancer.

Protocol Variations:

Various configurations of the assay can be performed. Four exemplaryprotocols are presented for the purposes of theoligonucleotide-sequencing assay. Samples can be any appropriatebiological sample. The protocols can be modified as desired. Forexample, the microvesicles can be isolated using alternate techniquesinstead or in addition to ultracentrifugation. Such techniques can bedisclosed herein, e.g., polymer precipitation (e.g., PEG), columnchromatography, and/or affinity isolation.

Protocol 1:

Ultracentrifugation of 1-5 ml bodily fluid samples (e.g.,plasma/serum/urine) (120K×g, no sucrose) with two washes of theprecipitate to isolate microvesicles.

Measure total protein concentration of recovered sample containing theisolated microvesicles.

Conjugate the isolated microvesicles to magnetic beads (for exampleMagPlex beads (Luminex Corp. Austin Tex.)).

Incubate conjugated microvesicles with oligonucleotide pool of interest.

Wash unbound oligonucleotides by retaining beads using magnet.

Elute oligonucleotides bound to the microvesicles.

Amplify and purify the eluted oligonucleotides.

Oligonucleotide sequencing (for example, Next generation methods; IonTorrent: fusion PCR, emulsion PCR, sequencing).

Assess oligonucleotide profile.

Protocol 2:

This alternate protocol does not include a microvesicle isolation step,microvesicles conjugation to the beads, or separate partitioning step.This may present non-specific binding of the oligonucleotides againstthe input sample.

Remove cells/debris from bodily fluid sample and dilute sample with PBScontaining MgCl₂ (2 mM).

Pre-mix sample prepared above with oligonucleotide library.

Ultracentrifugation of oligonucleotide/sample mixture (120K×g, nosucrose). Wash precipitated microvesicles.

Recover precipitate and elute oligonucleotides bound to microvesicles.

Amplify and purify the eluted oligonucleotides.

Oligonucleotide sequencing (for example, Next generation methods; IonTorrent: fusion PCR, emulsion PCR, sequencing).

Assess oligonucleotide profile.

Protocol 3:

This protocol uses filtration instead of ultracentrifugation and shouldrequire less time and sample volume.

Remove cells/debris from bodily fluid sample and dilute it with PBScontaining MgCl₂ (2 mM).

Pre-mix sample prepared above with oligonucleotide library.

Load sample into filter (i.e., 150K or 300K MWCO filter or any otherthat can eliminate unbound or unwanted oligonucleotides). Centrifugesample to concentrate. Concentrated sample should contain microvesicles.

Wash concentrate. Variant 1: Dilute concentrate with buffer specifiedabove to the original volume and repeat centrifugation. Variant 2:Dilute concentrate with buffer specified above to the original volumeand transfer concentrate to new filter unit and centrifuge. Repeattwice.

Recover concentrate and elute oligonucleotides bound to microvesicles.

Amplify and purify the eluted oligonucleotides.

Oligonucleotide sequencing (for example, Next generation methods; IonTorrent: fusion PCR, emulsion PCR, sequencing).

Assess oligonucleotide profile.

Protocol 4:

Ultracentrifugation of 1-5 ml bodily fluid sample (120K×g, no sucrose)with 2 washes of the precipitate to isolate microvesicles.

Pre-mix microvesicles with oligonucleotide pool.

Load sample into 300K MWCO filter unite and centrifuge (2000×g).Concentration rate is ˜3×.

Wash concentrate. Variant 1: Dilute concentrate with buffer specifiedabove to the original volume and centrifuge. Repeat twice. Variant 2:Dilute concentrate with buffer specified above to the original volumeand transfer concentrate to new filter unit and centrifuge. Repeat twice

Recover concentrate and elute oligonucleotides bound to microvesicles.

Amplify and purify the eluted oligonucleotides.

Oligonucleotide sequencing (for example, Next generation methods; IonTorrent: fusion PCR, emulsion PCR, sequencing).

Assess oligonucleotide profile.

In alterations of the above protocols, polymer precipitation is used toisolate microvesicles from the patient samples. For example, theoligonucleotides are added to the sample and then PEG4000 or PEG8000 at4% or 8% concentration is used to precipitate and thereby isolatemicrovesicles. Elution, recovery and sequence analysis continues asabove.

Example 6 Plasma/Serum Probing with an Oligonucleotide Probe Library

The following protocol is used to probe a plasma or serum sample usingan oligonucleotide probe library.

Input Oligonucleotide Library:

Use 2 ng input of oligonucleotide library per sample.

Input oligonucleotide library is a mixture of two libraries, cancer andnon-cancer enriched, concentration is 16.3 ng/ul.

Dilute to 0.2 ng/ul working stock using Aptamer Buffer (3 mM MgCl₂ in1×PBS)

Add 10 ul from working stock (equal to 2 ng library) to each optisealtube

Materials:

PBS, Hyclone SH30256.01, LN: AYG165629, bottle #8237, exp. July 2015

Round Bottom Centrifuge Tubes, Beckman 326820, LN:P91207

OptiSeal Centrifuge tubes and plugs, polyallomer Konical, Beckman361621, lot #Z10804SCA

Ultracentrifuge rotor: 50.4 TI

Ultracentrifuge rotor: 50.4 TI, Beckman Canis ID #0478

Protocol:

1 Pre-chill tabletop centrifuge, ultracentrifuge, buckets, and rotor at4° C.

2 Thaw plasma or serum samples

3 Dilute 1 ml of samples with 1:2 with Aptamer Buffer (3 mM MgCl₂ in1×PBS)

4 Spin at 2000×g, 30 min, 4° C. to remove debris (tabletop centrifuge)

5 Transfer supernatants for all samples to a round bottom conical

6 Spin at 12,000×g, 45 min, 4° C. in ultracentrifuge to removeadditional debris.

7 Transfer supernatant about 1.8 ml for all samples into new OptiSealbell top tubes (uniquely marked).

8 Add 2 ng (in 10 ul) of DNA Probing library to each optiseal tube

9 QS to 4.5 ml with Aptamer Buffer

10 Fix caps onto the OptiSeal bell top tubes

11 Apply Parafilm around caps to prevent leakage

12 Incubate plasma and oligonucleotide probe library for 1 hour at roomtemperature with rotation

13 Remove parafilm (but not caps)

14 Place correct spacer on top of each plugged tube

15 Mark pellet area on the tubes, insure this marking is facing outwardsfrom center.

16 Spin tubes at 120,000×g, 2 hr, 4° C. (inner row, 33,400 rpm) topellet microvesicles.

17 Check marking is still pointed away from center.

18 Completely remove supernatant from pellet, by collecting liquid fromopposite side of pellet marker and using a 10 ml syringe barrel and 21G2needle

19 Discard supernatant in appropriate biohazard waste container

20 Add 1 ml of 3 mM MgCl2 diluted with 1×PBS

21 Gentle vortex, 1600 rpm for 5 sec and incubate 5 min at RT.

22 QS to ˜4.5 mL with 3 mM Mg Cl2 diluted with 1×PBS

23 Fix caps onto the OptiSeal bell top tubes.

24 Place correct spacer on top of each plugged tube.

25 Mark pellet area on the tubes, insure this marking is facing outwardsfrom center.

26 Spin tubes at 120,000×g, 70 min, 4° C. (inner row 33,400 rpm) topellet microvesicles

27 Check marking in still pointed away from center.

28 Completely remove supernatant from pellet, by collecting liquid fromopposite side of pellet marker and using a 10 ml syringe barrel and 21G2needle

29 Discard supernatant in appropriate biohazard waste container

30 Add 1 ml of 3 mM MgCl2 diluted with 1×PBS

31 Gentle vortex, 1600 rpm for 5 sec and incubate 5 min at RT.

32 QS to ˜4.5 mL with 3 mM Mg Cl2 diluted with 1×PBS

33 Fix caps onto the OptiSeal bell top tubes.

34 Place correct spacer on top of each plugged tube.

35 Mark pellet area on the tubes, insure this marking is facing outwardsfrom center.

36 Spin tubes at 120,000×g, 70 min, 4° C. (inner row 33,400 rpm) topellet microvesicles

37 Check marking is still pointed away from center.

38 Save an aliquot of the supernatant (100 ul into a 1.5 ml tube)

39 Completely remove supernatant from pellet, by collecting liquid fromopposite side of pellet marker and using a 10 ml syringe barrel and 21G2needle

40 Add 50 ul of Rnase-free water to the side of the pellet

41 Leave for 15 min incubation on bench top

42 Cut top off tubes using clean scissors.

43 Resuspend pellet, pipette up and down on the pellet side

44 Measure the volume, make a note on the volume in order to normalizeall samples

45 Transfer the measured resuspended eluted microvesicles with boundoligonucleotides to a Rnase free 1.5 ml Eppendorf tube

46 Normalize all samples to 100 ul to keep it even across samples andbetween experiments.

Next Generation Sequencing Sample Preparation:

I) Use 50 ul of sample from above, resuspended in 100 ul H2O andcontaining microvesicle/oligo complexes, as template in Transposon PCR,14 cycles.

II) AMPure transposon PCR product, use entire recovery for indexing PCR,10 cycles.

III) Check indexing PCR product on gel, proceed with AMPure if band isvisible. Add 3 cylces if band is invisible, check on gel. Afterpurification quantify product with QuBit and proceed with denaturing anddiluting for loading on HiSeq flow cell (Illumina Inc., San Diego,Calif.).

IV) 5 samples will be multiplexed per one flow cell. 10 samples perHiSeq.

Example 7 Oligonucleotide Probe Library

This Example presents development of an oligonucleotide probe library todetect biological entities. In this Example, steps were taken to reducethe presence of double stranded oligonucleotides (dsDNA) when probingthe patient samples. The data were also generated comparing the effectsof 8% and 6% PEG used to precipitate microvesicles (and potentiallyother biological entities) from the patient samples.

Protocol:

1) Pre-chill tabletop centrifuge at 4° C.

2) Protease inhibition: dissolve 2 tablets of “cOmplete ULTRA MINIEDTA-free EASYpack” protease inhibitor in 1100 ul of H₂O (20× stock ofprotease inhibitor).

3) Add 50 ul of protease inhibitor to the sample (on top of frozenplasma) and start thawing: 1 ml total ea.

4) To remove cells/debris, spin samples at 10,000×g, 20 min, 4° C.Collect 1 ml supernatant (SN).

5) Mix 1 ml supernatant from step 4 with 1 ml of 2×PBS 6 mM MgCl₃,collect 400 ul into 3 tubes (replicates A, B, C) and use it in step 6.

6) Add competitor per Table 5: make dilutions in 1×PBS, 3 mM MgCl₂, mixwell, pour into trough, pipet using multichannel.

TABLE 5 Competitors Intermediate Volume from Buffer to make Type ofStock stock Number of stock to make intermediate Final Final unitsCompetitor Concentration concentration samples intermediate stock, ulstock Volume, ul Concentration ng/ul Salmon DNA — 40 — — — 425.5 0.8ng/ul tRNA — 40 — — — 425.5 0.8 x S1 20 0.5 280 65.5 2555.6 425.5 0.01

7) Incubate for 10 min, RT, end-over-end rotation

Pool of 6-3S and 8-3S oligonucleotide probing libraries is ready: 2.76ng/ul (˜185 ng). Save pool stock and dilutions. New pool can be made bymixing 171.2 ul (500 ng) of library 6-3S (2.92 ng/ul) with 190.8 ul (500ng) of library 8-3S (2.62 ng/ul). Aliquot pooled library into 30 ul andstore at −80 C.

Add ssDNA oligonucleotide probing library to the final concentration 2.5pg/ul for binding. Make dilutions in 1×PBS, 3 mM MgCl₂.

TABLE 6 Probe library calculations ul from Volume per Final OriginalRequired working original stock ul of buffer Final Number of sample fromconcentration stock, ng/ul Lib Name stock (ng/ul) to make working tomake working volume, ul samples working stock (pg/ul) 2.76 Pooled 0.126.1 694.1 720.2 60 10.9 2.5 library 6-3S/8-3S

8) Binding: Incubate for 1 h at RT with rotation.

9) Prepare polymer solution: 20% PEG8000 in 1×PBS 3 mM MgCl2 (dilute 40%PEG8000 with 2×PBS with 6 mM MgCl2). Add 20% PEG8000 to sample to thefinal concentration 6%. Invert few times to mix, incubate for 15 min at4 C

TABLE 7 PEG calculations PEG PEG Final Final Volume 20% PEG Volume ofbuffer to Sample volume Total Total 20% PEG MW stock, % conc., % volume,ul to add, ul adjust final volume, ul before adding PEG samples needed,ml 8000 20 6 622.8 186.9 −0.4 436.4 60 11.2

10) Spin at 10,000×g for 5 min, RT.

11) Remove SN, add 1 ml 1×PBS, 3 mM MgCl2 and wash pellet by gentleinvertion with 1 ml aptamer buffer.

12) Remove buffer, Re-suspend pellets in 100 ul H2O: incubate at RT for10 min on mixmate 900 rpm to re-suspend.

13) Make sure each sample is re-suspended by pipetting after step 13.Make notes on hardly re-suspendable samples.

14) 50 ul of re-suspended sample to indexing PCR→next generationsequencing (NGS).

15) Keep leftover at 4 C

Technical Validation:

The current protocol was tested versus a protocol using 8% PEG8000 toprecipitate microvesicles. The current protocol further comprises stepsto reduce dsDNA in the oligonucleotide probing libraries.

FIG. 5A shows the within sample variance (black) between bindingreplicates and the between sample variance (grey). Black is on top ofgrey, thus any observable grey oligo is informative about differences inthe biology of two patient samples. This evaluation of Sources ofVariance shows that the technical variances is significantly smallerthan the biological variance.

FIG. 5B shows the impact of using a higher proportion of single strandedDNA and PEG 6% isolation (white bars) compared to when there is a higheramount of double stranded DNA and 8% PEG (grey). This data indicatesthat the protocol in this Example improves biological separation betweenpatients.

The plots in FIG. 5C show the difference between an earlier protocol(PEG 8% with increased dsDNA) and a modified protocol of the Example(PEG 6% no dsDNA). The black is the scatter between replicates(independent binding events) and the grey is the difference betweenpatients. This data shows that the signal to noise increasedsignificantly using the newer protocol.

Patient Testing:

The protocol above was used to test patient samples having the followingcharacteristics:

TABLE 8 Patient characteristics Sample Type Description Cancer Mixedtype carcinoma; Malignant; Cancer Invasive, predominant intraductalcomponent (8500/3) Cancer Fibrocystic Changes; Invasive lobularcarcinoma - 8520/3; Lobular carcinoma in situ - 8520/2; Benign; In situand grade 3 intraepith; Malignant; Fat necrosis, periductalinflammation, malignant cellsFat necrosis; Inflammation; Benign; CancerInvasive, predominant intraductal component (8500/3) Cancer Mucinous(colloid) adenocarcinoma (8480/3) Cancer Invasive lobular carcinoma -8520/3; Microcalcifications; Benign; Malignant; Cancer OtherfibrocysticchangeInvasive, NOS (8500/3) Cancer Invasive ductal carcinoma, nototherwise specified (NOS) - 8500/3; Malignant; Cancer Invasive ductalcarcinoma, not otherwise specified (NOS) - 8500/3; Malignant; CancerIntraductal carcinoma, non-infiltrating, NOS (in situ) (8500/2) CancerAtypical lobular hyperplasia Otherfibrocystic changes, inter andintralobular fibrosis, apocrine metaplasia, columnar cell change,microcalcificationsInvasive, NOS (8500/3) Cancer FibroadenomaInvasive,NOS (8500/3) Cancer Ductal carcinoma in situ - 8500/2; Invasive ductalcarcinoma, not otherwise specified (NOS) - 8500/3; Microcalcifications;Benign; In situ and grade 3 intraepith; Malignant; Cancer Ductalcarcinoma in situ - 8500/2; Invasive lobular carcinoma - 8520/3; Lobularcarcinoma in situ - 8520/2; In situ and grade 3 intraepith; Malignant;Cancer Ductal carcinoma in situ - 8500/2; Invasive ductal carcinoma, nototherwise specified (NOS) - 8500/3; Microcalcifications; Benign; In situand grade 3 intraepith; Malignant; Focal Micropapillary Features,invasive ductal carcinoma with micropapillary features, invasive ductalcarcinoma with mucinous and micropapillary featInvasive ductal carcinomawith micropapillary and mucinous features; Invasive micropapillarycarcinoma - 8507/3; Malignant; Cancer Invasive, predominant intraductalcomponent (8500/3) Cancer Invasive ductal carcinoma, not otherwisespecified (NOS) - 8500/3; Malignant; Cancer Invasive, NOS (8500/3)Cancer Infiltrating duct and lobular carcinoma (8522/3) Cancer Invasive,predominant in situ component (8522/3) Non-Cancer Otherusual ductalhyperplasia, apocrine metaplasia, microcysts, elastosis Non-CancerOtherstromal fibrosis, fibrous cyst wall Non-Cancer Otherfibrocysticchange, stromal fibrosis, cyst formation, microcalcifications, apocrinemetaplasia, sclerosing adenosis, usual ductal hyperplasia Non-CancerOtherfibrocystic changes, apocrine metaplasia, cystic change, usualductal hyperplasia Non-Cancer Otherfibrocystic change,microcalcifications Non-Cancer Fibroadenoma Non-Cancer Otherintraductalpapilloma, sclerosis, microcalcifications, stromal fibrosis Non-CancerFibroadenoma Non-Cancer Otherfat necrosis Non-Cancer Otherstromalfibrosis, microcalcifications Non-Cancer Otherfibrocystic change,microcystic change, focal secretory features Non-Cancer Otherstromalfibrosis Non-Cancer Fibroadenoma Otheradenosis, columnar cellchange/hyperplasia, usual ductal hyperplasia Non-Cancer OtherFNA -insufficient material for diagnosis Non-Cancer Otherintraductalpapilloma Non-Cancer Otherfibrocystic changes, duct ectasia, usualductal hyperplasia, apocrine metaplasia, microcalcifications

Microvesicles (and potentially other biological entities) wereprecipitated in blood (plasma) samples from the above patients usingpolymer precipitation with PEG as indicated above. The protocol was usedto probe the samples with the oligonucleotide probe libraries. Sequencesthat bound the PEG precipitated samples were identified using nextgeneration sequencing (NGS).

FIG. 5D shows scatter plots of a selection of results from testing the40 patients listed previously. The spread in the data indicates thatlarge numbers of oligos were detected that differed between samples. Thenumber of significant oligos found is much greater than would beexpected randomly as shown in Table 9. The table shows the number ofoligonucleotides sorted by copy number detected and p-value. The d-#indicates the number copies of a sequence observed for the data in therows.

TABLE 9 Expected versus observed sequences Total Number P-0.1 P-0.05P-0.01 P-0.005 d-50 83,632 47,020 30,843 5,934 2,471 d-100 52,647 29,10619,446 3,893 1,615 d-200 28,753 14,681 9,880 2,189 914 d-500 10,1554,342 2,927 725 315 d-50 100.0% 56.2% 36.9% 7.1% 3.0% d-100 100.0% 55.3%36.9% 7.4% 3.1% d-200 100.0% 51.1% 34.4% 7.6% 3.2% d-500 100.0% 42.8%28.8% 7.1% 3.1% Maximum expected 10.0% 5.0% 1.0% 0.5%

As a control, the cancer and non-cancer samples were randomly dividedinto two groups. Such randomization of the samples significantly reducedthe number of oligos found that differentiate between sample groups.Indeed, there was a 50-fold increase in informative oligos between thecancer/non-cancer grouping versus random grouping. FIG. 5E shows data asin Table 9 and indicates the number of observed informative oligosbetween the indicated sample groups.

FIG. 5F shows distinct groups of oligos that differentiate betweencancer and non-cancer samples. The figure shows a heatmap of the 40samples tested with oligos selected that had more than 500 copies andp-value less than 0.005. There are clear subpopulations emerging with adistinct non-cancer cohort at the top. The non-cancer samples have boxesaround them on the left axis. FIG. 5G is similar and shows results withan additional 20 cancer and 20 non-cancer samples. As shown, analysiswith the 80 samples provides the emergence of more distinct and largerclusters.

The data for the additional 80 samples was also used to compare theconsistency of informative oligos identified in different screeningexperiments. Of the 315 informative oligos identified using the firstset of 40 patients, 86% of them showed fold-change in a consistentmanner when tested on the independent set of 40 patients.

Example 8 Enrichment of Oligonucleotide Probes Using a Balanced LibraryDesign

In this Example, a naïve ADAPT oligonucleotide library was screened toenrich oligonucleotides that identify microvesicles circulating in theblood of breast cancer patients and microvesicles circulating in theblood of healthy, control individuals (i.e., without breast cancer). Theinput library was the naïve F-TRin-35n-B 8-3s library, which comprises a5′ region (5′ CTAGCATGACTGCAGTACGT (SEQ ID NO. 4)) followed by therandom naïve aptamer sequences of 35 nucleotides and a 3′ region (5′CTGTCTCTTATACACATCTGACGCTGCCGACGA (SEQ ID NO. 5)). The “balanced” designis described in Example 23 of Int'l Patent Publication WO/2015/031694(Appl. No. PCT/US2014/053306, filed Aug. 28, 2014), which isincorporated by reference herein in its entirety. The working librarycomprised approximately 2×10¹³ synthetic oligonucleotide sequences. Thenaïve library may be referred to as the “L0 Library” herein.

The L0 Library was enriched against fractionated plasma samples frombreast cancer patients and from healthy (non-breast cancer) controlsusing the protocol shown in FIG. 12A. In Step 1, an aliquot ofapproximately 10¹¹ sequences of PCR-amplified L0 was incubated withpooled blood-plasma from 59 breast cancer patients with positive biopsy(represented by “Source A” in FIG. 12A). In parallel, another aliquot of10¹¹ sequences was incubated with pooled blood-plasma from 30 patientswith suspected breast cancer who proved negative on biopsy and 30 selfdeclared healthy women (represented by “Source B” in FIG. 12A). In Step2, microvesicles (extracellular vesicles, “EV”) were precipitated usingultracentrifugation (UC) from both L0-samples. The EV-associatedoligodeoxynucleotides (ODNs) were recovered from the respective pellets.In Step 3, a counter-selection step (Step 3) was carried out byincubation of each enriched library with plasma from the differentcohorts to drive the selection pressure towards enrichment of ODNsspecifically associated with each sample cohort. In this step, sequencescontained in the EV pellets were discarded. In Step 4, a second positiveselection was performed. In this step, the sequences contained in therespective supernatants (sn) from Step 3 were mixed with plasma fromanother aliquot of each positive control sample-population, and EVs wereagain isolated. EV-associated ODNs were recovered, representing twosingle-round libraries called library L1 for positive enrichment ofcancer (positive biopsy) patients, and library L2 for the positiveenrichment against control patients. In a final step, L1 and L2 wereamplified by PCR, reverted to single stranded DNA (ssDNA), and mixed toyield library L3.

This enrichment scheme was iterated two times more using L3 as the inputto further reduce the complexity of the profiling library toapproximately 10⁶ different sequences. In Step 2, UC was used forpartitioning of microvesicles, which may increase the specificity forthe EV fraction. In Steps 3 and 4, partitioning was performed usingPEG-precipitation. This procedure enriches for ODNs specific for eachbiological source. Library L3 contains those ODNs that are associatedwith targets characteristic for EV-populations from both sources, i.e.ODNs acting as aptamers that bind to molecules preferentially expressedin each source. A total of biopsy-positive (n=59), biopsy-negative(n=30), and self-declared normal (n=30) were used in the first round ofL3 enrichment, while only the cancer and non-cancer samples were used inthe subsequent rounds.

The enriched libraries were characterized usingnext-generation-sequencing (NGS) to measure copy numbers of sequencescontained in each profiling library. NGS of L0 shows that the vastmajority of sequences existed in low copy numbers, whereas libraries L1and L2 showed significantly higher average counts per sequence (FIG.12B) and a reduced amount of different sequences, with unaltered totalvalid reads, (FIG. 12C) consistent with an enrichment process.

Example 9 Analysis of ADAPT-Identified Biomarkers

As described herein, e.g., in the section entitled “Aptamer TargetIdentification,” an unknown target recognized by an aptamer can beidentified. In this Example, an oligonucleotide probe library (alsoreferred to as Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT)libraries or Topographical Oligonucleotide Probe “TOP” libraries) wasdeveloped as described here and targets of the screened oligonucleotideswere determined. This Example used a ADAPT library generated byenriching microvesicles collected from the blood of breast cancerpatients and normal controls (i.e., non-cancer individuals). Theenrichment protocols are described herein in Example 8.

Materials & Methods

SBED Library Conjugation

A naïve F-TRin-35n-B 8-3s library was enriched against microvesiclesfrom normal female plasma. The naïve unenriched library comprised a 5′region (5′ CTAGCATGACTGCAGTACGT (SEQ ID NO. 4)) followed by the randomnaïve aptamer sequences of 35 nucleotides and a 3′ region (5CTGTCTCTTATACACATCTGACGCTGCCGACGA (SEQ ID NO. 5)). The naïve library maybe referred to as the “L0 Library” herein and the enriched libraryreferred to as the “L2 library.” See Example 8. The screened library wasPCR amplified with a C6-amine sense primer (C6 Amine-5′CTAGCATGACTGCAGTACGT 3′ (SEQ ID NO. 4)) and a 5′ phosphorylatedanti-sense primer (5′ Phos TCGTCGGCAGCGTCA (SEQ ID NO. 6)), the purifiedproduct was strand separated and conjugated with sulfo-SBED (ThermoScientific) according to Vinkenborg et al. (Angew Chem Int Ed Engl.2012, 51:9176-80) with the following modifications: The reaction wasscaled down to 5 μg C6-amine DNA library (8.6 μM) in 25 mM HEPES-KOH,0.1M NaCl, pH 8.3 and incubated with either 100-fold molar excess ofsulfo-SBED or DMSO in a 21 μL volume for 30 min at room temp in thedark. The SBED-conjugated library was immediately separated from theunconjugated library and free sulfo-SBED by injection onto a WatersX-Bridge™ OST C-18 column (4.6 mm×50 mm) and fractionated by HPLC(Agilent 1260 Infinity) with a linear gradient Buffer A: 100 mM TEAA,pH7.0, 0% ACN to 100 mM TEAA, pH7.0, 25% ACN at 0.2 ml/min, 65° C. ThereSBED-conjugated fractions were desalted into water with Glen Gel-Pak™Cartridges and concentrated by speed-vac. SBED conjugation was confirmedby LC-MS and/or a dot blot with streptavidin-HRP detection.

Binding Reaction and Cross-Linking

SBED library functionalization was tested by performing the ADAPT assaywith SBED vs DMSO mock conjugated control C6-amine library and sequencedon a HiSeq 2500™ (Illumina Corp.). The aptamer precipitation wasperformed with forty-eight ADAPT reactions incubated for 1 hr withend-over-end rotation at room temp with a 5 ng input of SBED conjugatedlibrary per 200 μL of plasma (pre-spun to remove cellular debris at10,000×g for 20 min, 4° C.) in 1×PBS, 3 mM MgCl₂, 0.01 mM dextransulfate, 40 ng/μl salmon sperm DNA and 40 ng/μl yeast transfer RNA, andcOmplete ULTRA Mini EDTA-Free™ protease inhibitors (Roche) equivalent to˜240 ng library and 9.6 mls plasma. A duplicate set of 48 reactions wasprepared with the DMSO control C6-amine library. Aptamer library-proteincomplexes were precipitated with incubation in 6% PEG8000 for 15 min at4° C. then centrifuged at 10,000×g for 5 min. Pellets were washed with 1ml 1×PBS, 3 mM MgCl2 by gentle inversion to remove unbound aptamers. Thewashed pellets were resuspended in 100 μL of water and subjected tophoto-cross-linking at 365 nm with a hand-held 3UV (254 NM/302 NM/365NM) lamp, 115 volts (Thermo Scientific) for 10 min on ice with 1-2 cmbetween the 96-well plate and lamp.

Oligonucleotide Precipitation

Cross-linked reactions were subsequently pooled (˜4.8 ml) per library or4.8 ml of 1×PBS (AP bead only control) and incubated with 10 μL ofPrepared Dynabeads® My Oile™ Streptavidin C1 (10 mg/ml) (LifeTechnologies) (pre-washed with 1×PBS, 0.01% Triton X-100) shaking for 1hr at room temp. Beads were transferred to an eppendorf tube and lysedfor 20 min with lysis buffer (50 mM Tris-HCl, 10 mM MgCl2, 200 mM NaCl,0.5% Triton X-100, 5% glycerol, pH 7.5) on ice, washed 3 times with washbuffer 1 (10 mM Tris-HCl, 1 mM EDTA, 2M NaCl, 1% Triton X-100), followedby 2 times with wash buffer 2 (10 mM Tris-HCl, 1 mM EDTA, 2M NaCl, 0.01%Triton X-100) as described by Vinkenborg et al. (Angew Chem Int Ed Engl.2012, 51:9176-80). Cross-linked proteins were eluted by boiling 15 minin 1×LDS sample buffer with reducing agent added (Life Technologies) andloaded on a 4-12% SDS-PAGE gradient gel (Life Technology). Proteins andDNA were detected with double staining with Imperial Blue Protein Stain(Thermo Scientific) followed by Prot-SIL2™ silver stain kit (Sigma) usedaccording to manufacturer's instructions in order to enhance sensitivityand reduce background.

Protein Identification

Protein bands that appeared to differ between the cancer and normal wereexcised from the gradient gels and subjected to liquidchromatography-tandem mass spectrometry (LC-MS/MS).

Results

ADAPT protein targets were identified from bands cut from a silverstained SDS-PAGE gel (FIG. 6). Aptamer-SBED protein complexes (lane 3)or Aptamer-DMSO protein complexes (control-lane 4) were precipitatedwith 6% PEG8000, subjected to UV photo-cross-linking, and pulled-downwith Streptavidin coated beads. Eluate was analyzed under reducingconditions by SDS-PAGE and silver staining Aptamer library alone (5 ng)(lane 1) was loaded as a control for migration of the library (second tobottom arrows) and an equal volume of eluate from a bead only sample(lane 4) was loaded as a streptavidin control to control for potentialleaching of the streptavidin monomer (bottom arrow) under the harshelution conditions. Upper arrows (“Targets”) indicate specific or morepredominant bands identified with the SBED-conjugated library vs. themock DMSO treated control C6-amine library. Indicated target proteinbands were cut out and sent for LC-MS/MS protein identification orindicated DNA library bands were eluted, reamplified and sequenced. Theidentified proteins are those that appeared as upregulated in the normalsamples.

Tables 10-17 list human proteins that were identified in 8 bands excisedfrom the silver stained gel. In all tables the proteins are thoseidentified in the oligo-SBED protein complexes with proteins identifiedin the corresponding control lanes removed. The band numbers in thetables indicate different bands cut from the gel (FIG. 6). Accessionnumbers in the table are from the UniProt database (www.uniprot.org).“GN=” is followed by the gene name. Various protein classificationsindicated in the Tables 10-17 include Nucleic Acid Binding Proteins(NAB), Tumor suppressors (TS), cell adhesion/cytoskeletal (CA/CK) andabundant plasma proteins (ABP).

TABLE 10 Band 3 Accession number Class Protein name P02538 CA/CKKeratin, type II cytoskeletal 6A GN = KRT6A P15924 CA/CK Desmoplakin GN= DSP P04259 CA/CK Keratin, type II cytoskeletal 6B GN = KRT6B P60709CA/CK Actin, cytoplasmic 1 GN = ACTB P20930 CA/CK Filaggrin GN = FLGP07476 CA/CK Involucrin GN = IVL P31947 TS 14-3-3 protein sigma GN = SFNQ7Z794 CA/CK Keratin, type II cytoskeletal 1b GN = KRT77 P02545 NABPrelamin-A/C GN = LMNA P19012 CA/CK Keratin, type I cytoskeletal 15 GN =KRT15 P47929 CA/CK & TS Galectin-7 GN = LGALS7 P11142 Heat shock cognate71 kDa protein GN = HSPA8 P58107 NAB Epiplakin GN = EPPK1 P08107 Heatshock 70 kDa protein 1A/1B GN = HSPA1A Q02413 CA/CK Desmoglein-1 GN =DSG1 P06396 CA/CK Gelsolin GN = GSN O60814 NAB Histone H2B type 1-K GN =HIST1H2BK P68104 NAB Elongation factor 1-alpha 1 GN = EEF1A1 P05387 NAB60S acidic ribosomal protein P2 GN = RPLP2 Q7RTS7 CA/CK Keratin, type IIcytoskeletal 74 GN = KRT74 P31946 TS 14-3-3 protein beta/alpha GN =YWHAB Q13835 CA/CK Plakophilin-1 GN = PKP1 P14923 CA/CK functionplakoglobin GN = JUP P09651 NAB Heterogeneous nuclear ribonucleoproteinA1 GN = HNRNPA1 P07900 Heat shock protein HSP 90-alpha GN = HSP90AA1Q96KK5 NAB Histone H2A type 1-H GN = HIST1H2AH P04406- CA/CKGlyceraldehyde-3-phosphate dehydrogenase GN = GAPDH P10412 NAB HistoneH1.4 GN = HIST1H1E P04792 Heat shock protein beta-1 GN = HSPB1 Q9NZT1Calmodulin-like protein 5 GN = CALML5 P81605 Dermcidin GN = DCD P27348TS 14-3-3 protein theta GN = YWHAQ P55072 NAB Transitional endoplasmicreticulum ATPase GN = VCP Q09666 NAB Neuroblastdifferentiation-associated protein AHNAK GN = AHNAK P23246 NAB Splicingfactor, proline- and glutamine-rich GN = SFPQ Q15149 CA/CK Plectin GN =PLEC Q8NC51 NAB Plasminogen activator inhibitor 1 RNA-binding protein GN= SERBP1 P07237 Protein disulfide-isomerase GN = P4HB O60437 CA/CKPeriplakin GN = PPL P01717 ABP Ig lambda chain V-IV region Hil P55884NAB Eukaryotic translation initiation factor 3 subunit B GN = EIF3BP11021 78 kDa glucose-regulated protein GN = HSPA5 P01024 Complement C3GN = C3 P04350 CA/CK Tubulin beta-4A chain GN = TUBB4A P01857 ABP Iggamma-1 chain C region GN = IGHG1 P61247 NAB 40S ribosomal protein S3aGN = RPS3A P62937 Peptidyl-prolyl cis-trans isomerase A GN = PPIA O15020CA/CK Spectrin beta chain, non-erythrocytic 2 GN = SPTBN2 P30101 Proteindisulfide-isomerase A3 GN = PDIA3 Q6KB66 CA/CK Keratin, type IIcytoskeletal 80 GN = KRT80 Q9UJU6 CA/CK Drebrin-like protein GN = DBNLP47914 NAB 60S ribosomal protein L29 GN = RPL29 P39023 NAB 60S ribosomalprotein L3 GN = RPL3 A6NMY6 CA/CK Putative annexin A2-like protein GN =ANXA2P2 P60174 CA/CK Triosephosphate isomerase GN = TPI1 P35241 CA/CKRadixin GN = RDX P07305 NAB Histone H1.0 GN = H1F0 P15259 CA/CKPhosphoglycerate mutase 2 GN = PGAM2 P0CG05 ABP Ig lambda-2 chain Cregions GN = IGLC2 Q92817 CA/CK Envoplakin GN = EVPL P06733 NAB MBP-1 ofAlpha-enolase GN = ENO1 P22626 NAB Heterogeneous nuclearribonucleoproteins A2/B1 GN = HNRNPA2B1 P62424 NAB 60S ribosomal proteinL7a GN = RPL7A P60660 CA/CK Myosin light polypeptide 6 GN = MYL6 P04083NAB Annexin A1 GN = ANXA1 Q14134 NAB Tripartite motif-containing protein29 GN = TRIM29 P39019 NAB 40S ribosomal protein S19 GN = RPS19 Q8WVV4CA/CK Protein POF1B GN = POF1B Q02878 NAB 60S ribosomal protein L6 GN =RPL6 Q9Y6X9 NAB MORC family CW-type zinc finger protein 2 GN = MORC2Q9NQC3 NAB Reticulon-4 GN = RTN4 Q5T753 CA/CK Late cornified envelopeprotein 1E GN = CA/CK E

TABLE 11 Band 9 Accession number Class Protein name P61626 Lysozyme C GN= LYZ Q9HCK1 NAB DBF4-type zinc finger-containing protein 2 GN = ZDBF2

TABLE 12 Band 1 Accession number Class Protein name P01834 ABP Ig kappachain C region GN = IGKC P01765 ABP Ig heavy chain V-III region TILP04003 NAB C4b-binding protein alpha chain GN = C4BPA P60709 CA/CKActin, cytoplasmic 1 GN = ACTB Q5T751 CA/CK Late cornified envelopeprotein 1C GN = LCE1C

TABLE 13 Band 5 Accession number Class Protein name P01860 ABP Iggamma-3 chain C region GN = IGHG3 O60902 NAB Short stature homeoboxprotein 2 GN = SHOX2

TABLE 14 Band 7 Accession number Class Protein name Q04695 CA/CKKeratin, type I cytoskeletal 17 GN = KRT17 Q7Z794 CA/CK Keratin, type IIcytoskeletal 1b GN = KRT77 Q6KB66 CA/CK Keratin, type II cytoskeletal 80GN = KRT80 P01833 Polymeric immunoglobulin receptor GN = PIGR P01042Kininogen-1 GN = KNG1 Q02413 CA/CK Desmoglein-1 GN = DSG1 P15924 CA/CKDesmoplakin GN = DSP Q8TF72 Protein Shroom3 GN = SHROOM3 P02671 ABPFibrinogen alpha chain GN = FGA Q5T749 CA/CK Keratinocyte proline-richprotein GN = KPRP Q5VZP5 Inactive dual specificity phosphatase 27 GN =DUSP27 Q5T751 CA/CK Late cornified envelope protein 1C GN = LCE1C Q9UL12Sarcosine dehydrogenase, mitochondrial GN = SARDH P00698 Lysozyme C OS =Gallus gallus GN = LYZ Q8N114 Protein shisa-5 GN = SHISA5

TABLE 15 Band 15 Accession number Class Protein name P08238 Heat shockprotein HSP 90-beta GN = HSP90AB1 P68104 NAB Elongation factor 1-alpha 1GN = EEF1A1 P02675 ABP Fibrinogen beta chain GN = FGB Q8TF72 ProteinShroom3 GN = SHROOM3 P0CG05 ABP Ig lambda-2 chain C regions GN = IGLC2P78386 CA/CK Keratin, type II cuticular Hb5 GN = KRT85 Q7Z5Y6 Bonemorphogenetic protein 8A GN = BMP8A O14633 CA/CK Late cornified envelopeprotein 2B GN = LCE2B

TABLE 16 Band 17 Accession number Class Protein name P02538 CA/CKKeratin, type II cytoskeletal 6A GN = KRT6A P01834 ABP Ig kappa chain Cregion GN = IGKC P06702 Protein S100-A9 GN = S100A9 P68104 NABElongation factor 1-alpha 1 GN = EEF1A1 P01024 Complement C3 GN = C3P81605 Dermcidin GN = DCD P05109 Protein S100-A8 GN = S100A8 Q5T751CA/CK Late cornified envelope protein 1C GN = LCE1C

TABLE 17 Band 19 Accession number Class Protein name P02768 NAB Serumalbumin GN = ALB P0CG05 ABP Ig lambda-2 chain C regions GN = IGLC2P06702 Protein S100-A9 GN = S100A9 P08238 Heat shock protein HSP 90-betaGN = HSP90AB1 P60709 CA/CK Actin, cytoplasmic 1 GN = ACTB P13647 CA/CKKeratin, type II cytoskeletal 5 GN = KRT5 P01616 ABP Ig kappa chain V-IIregion MIL Q86YZ3 CA/CK Homerin GN = HRNR P01857 ABP Ig gamma-1 chain Cregion GN = IGHG1 P62805 NAB Histone H4 GN = HIST1H4A P59665 Neutrophildefensin 1 GN = DEFA1 P61626 Lysozyme C GN = LYZ P01024 ABP ComplementC3 GN = C3 Q8TF72 Protein Shroom3 GN = SHROOM3 P83593 ABP Ig kappa chainV-IV region STH (Fragment) P01700 ABP Ig lambda chain V-I region HAP01877 ABP Ig alpha-2 chain C region GN = IGHA2 Q9UL12 Sarcosinedehydrogenase, mitochondrial GN = SARDH Q6NXT2 NAB Histone H3.3C GN =H3F3C P02788 NAB Lactotransferrin GN = LTF P02787 ABP Serotransferrin GN= TF

Certain proteins were identified in multiple bands. For example, IGLC2was identified in bands 3, 15 and 19 and SHROOM3 was identified in bands7, 15, 19. This may be due to degradation products, isoforms or thelike. These experiments identified 108 proteins (plus 2 lysozymecontrols), comprising among others 34 Nucleic Acid Binding Proteins(NAB) where 7 of the 34 are putative tumor suppressors/repressors; 37cell adhesion/cytoskeletal (CA/CK); and 14 abundant plasma proteins(ABP). All of the tumor suppressors/repressors are DNA/RNA bindingproteins. Other proteins comprise chaperones, signaling molecules etc.

The biomarkers in this Example can be used to detect microvesicles thatare indicative of cancer or non-cancer samples.

Example 10 Identification of Biomarkers Through Affinity Enrichment withan Enriched Oligonucleotide Library and Mass Spectrometry

This Example continues upon the Example above. Identification ofprotein-protein and nucleic acid-protein complexes by affinitypurification mass spectrometry (AP-MS) can be hampered in samplescomprising complex mixtures of biological components (e.g., bodilyfluids including without limitation blood and derivatives thereof). Forexample, it may be desirable to detect low abundance protein and nucleicacid-protein complexes in a complex milieu comprising various componentsthat may interact promiscuously with specific binding sites such as highabundance proteins that interact non-specifically with the affinityresin. AP-MS has been used previously to enrich for pre-identifiedtargets of interest using individual DNA or RNA aptamers or specificnucleic acid binding domains. In this Example, an enrichedoligonucleotide probing library was used as the affinity reagent. Thisapproach combined with mass spectrometry enables the identification ofdifferentially expressed biomarker from different disease states orcellular perturbations without relying on a priori knowledge of thetargets of interest. Such biomarker may comprise proteins, nucleicacids, miRNA, mRNA, carbohydrates, lipid targets, combinations thereof,or other components in a biological system.

The method comprises identification of an enriched oligonucleotide probelibrary according to the methods of the invention followed by targetidentification with affinity purification of the bound probing libraryand mass spectrometry. The members of the enriched oligonucleotideprobing library comprise an affinity tag. A biological sample is probedwith the oligonucleotide probe library, affinity purification of theoligonucleotide probe library via the affinity tag is performed whichwill accordingly purify biological entities in complex with variousmembers of the probe library, and read-out of targets that purified withthe members of the probe library is performed using liquidchromatography-tandem mass spectrometry (LC-MS/MS) for proteins oroligonucleotide targets (e.g., miRNA or mRNA) with next generationsequencing (NGS). Confirmation of protein targets is performed usingquantitative mass spectrometry (MS), e.g., using MRM/SRM or SWATH basedmethods.

The method of the Example lends itself to various options. For example,any appropriate affinity tags can be used for affinity pull-down,including without limitation anti-sense oligonucleotides, biotin,polyhistidine, FLAG octapeptide (i.e., N-DYKDDDDK-C (SEQ ID NO. 7),where N stands for Amino-terminus and C stands for Carboxy terminus),3×FLAG, Human influenza hemagglutinin (HA)-tag (i.e., N-YPYDVPDYA-C (SEQID NO. 8)), myc-tag (N-EQKLISEEDL-C (SEQ ID NO. 9)), other such as knownin the art, and combinations thereof. Similarly, any appropriateenrichment support can be used in addition to the magnetic streptavidinbeads exemplified herein, including without limitation other beadsystems, agarose beads, planar arrays or column chromatography supports.It follows that the various supports can be coupled with the variousaffinity reagents appropriate for the oligonucleotide library, includingwithout limitation streptavidin, avidin, anti-His tag antibodies,nickel, and the like. The different affinity tags and supports can becombined as desired. This Example used cross-linking but in certaincases such cross-linking is not necessary and may even be undesirable,e.g., to favor identification of high affinity complex formation. Whencross-linking is desired, any appropriate cross-linkers can be used tocarry out the invention, including BS2G, DSS, formaldehyde, and thelike. Other appropriate cross-linkers and methods are described herein.See, e.g., Section “Aptamer Target Identification.” Lysis buffers andwash stringencies can be varied, e.g, depending on whether complexes arecross-linked or not. Less stringent lysis/wash conditions may produce awider array of potential protein complexes of interest whereas morestringent lysis/wash conditions may favor higher affinity oligo-targetcomplexes and/or targets comprising specific proteins (e.g., bydisassociating larger complexes bound to the oligos). One of skill willfurther appreciate that qualitative and/or quantitative LC-MS/MS may beused for target detection and verification. Similarly, metaboliclabeling and label-free approaches may be used for quantitative MS,including without limitation spectral counting, SILAC, dimethyllabeling, TMT labeling, Targeted MS with SRM/MRM or SWATH, and the like.

References

-   Vickenborg et al. “Aptamer based affinity labeling of proteins”,    Angew Chem Int. 51(36):9176-80 (2012).-   Tacheny, M, Arnould, T., Renard, A. “Mass spectrometry-based    identification of proteins interacting with nucleic acids”, Journal    of Proteomics 94; 89-109 (2013).-   Faoro C and Ataide S F. “Ribonomic approaches to study the    RNA-binding proteome.”, FEBS Lett. 588(20):3649-64 (2014).-   Budayeva H G, Cristea, I M, “A mass spectrometry view of stable and    transient protein interactions.” Adv Exp Med Biol. 806:263-82    (2014).

Example 11 Protocol for Affinity Capture Using Oligonucleotide ProbingLibrary

This Example presents a detailed protocol for the method of affinitycapture using an oligonucleotide probing library presented in theExample above.

Protocol:

The oligonucleotide probe library comprises F-TRin-35n-B-8-3s describedherein either desthiobiotin labeled or unlabeled library and binding tonormal (i.e., non-cancer) female plasma. The oligonucleotide probelibrary is enriched against the plasma samples as described elsewhere(e.g., in Example 7). The plasma samples are processed separatelyagainst the desthiobiotin labeled or unlabeled oligonucleotidelibraries. General parameters included the following:

48 normal plasma samples are pooled for enrichment of eacholigonucleotide library (Desthiobiotin or Unlabeled)

200 μl input plasma per sample

Ultracentrifugation (UC) is used to pre-clear the samples

5 ng of each aptamer library is added to each sample

Binding competitors for all library samples include 0.01×Si (dextransulfate), 340 ng for tRNA and 340 ng Salmon sperm DNA as describedelsewhere herein

6% PEG 8000 is used for precipitation of microvesicles within thesamples

Affinity purification is performed with C1 Streptavidin beads (MyOneStrptavidin Beads C1-65001, lot 2 ml (10 mg/ml))

Buffers:

Plasma dilution: 6 mM MgCl2 in 2×PBS

Pellet Wash Buffer: 1×PBS, 3 mM MgCl2

PEG Ppt Buffer: 20% Peg8000 in 1×PBS, 3 mM MgCl2

Bead Prep Buffer: 1×PBS containing 0.01% Triton X-100

Lysis Buffer: prepare a 2× stock solution consisting of 100 mM Tris-HCl,20 mM MgCl2, 400 mM NaCl, 1% Triton X-100, 10% glycerol, pH 7.5. Dilutedto 1× with water 1:1 prior to using.

AP Wash buffer 1: 10 mM Tris-HCl, 1 mM EDTA, 2M NaCl, 1% Triton X-100,pH 7.5

AP wash buffer 2: 10 mM Tris-HCL, 1 mM EDTA, 2M NaCl, 0.01% TritonX-100, pH 7.5

Biotin Elution buffer 1: 5 mM Biotin, 20 mM Tris, 50 mM NaCl, pH 7.5

1×LDS, 1× Reducing buffer 2

Reagent/Instrument Prep:

Pre-chill Ultracentrifuge to 4° C.

Protease inhibition: dissolve 2 tablets of “cOmplete ULTRA MINIEDTA-free EASYpack” protease inhibitor in 1100 μl of H2O (20× stock ofprotease inhibitor).

Plasma Preparation (for each of Desthiobiotin or UnlabeledOligonucleotide Libraries):

1. Add 50 μl of protease inhibitor to each ml of sample (on top offrozen plasma) in a room temperature (RT) water bath. Will use 20 mls ofpooled plasma, so 1100 μl inhibitor.

2. To remove cell/debris, spin samples at 7500×g 20 min, 4° C. in theUltracentrifuge.

3. Collect the supernatant, pool and measure volume & record.

4. Add an equal volume of 2×PBS, 6 mM MgCl₂ to the plasma.

5. Label low-retention eppendorf tubes 1-96.

6. Transfer 400 μl of each sample to eppendorf tubes based onappropriate tube map

7. Using an electronic P200, add competitors: 8.6 μl of 40 ng/μl Salmonsperm DNA; 8.6 μl of 40 ng/μl tRNA; 8.6 μl of 0.5× S1.

8. Incubate at RT with end over end rotation for 10 min.

9. Add 10 μL of appropriate oligo library, mix well. Save any leftoverdiluted library for gel control (see below).

10. Incubate 1 hr at RT with end over end rotation.

11. Using an electronic repeat P100, add 187 μl of 20% PEG 8000 tosample for a final 6% concentration to the 435.5 μl of sample/oligolibrary. Invert a few times to mix and incubate for 15 min at 4° C.

12. Spin each sample in table top centrifuge at 10,000×g for 5 min.

13. Remove supernatant and discard, add 1 ml 1×PBS, 3 mM MgCl₂ topellet.

14. Wash pellet by gentle inversion

15. Remove buffer, re-suspend pellets in 100 μl 1×PBS, 3 mM MgCl₂:incubate at RT for 10 min on mixmate@900 rpm to re-suspend. Make sureeach sample is well re-suspended by pipetting.

16. Pool all desthiobiotin library samples into one 50 ml falcon tube,and the unlabeled library into another, total volume for each should be4800 μl.

17. Take 10 μL aliquot for the input into AP sample for gel (add 10 μLof 2×LDS buffer w/2× reducing agent.

Affinity Purification:

18. Prepare 10 μL of MyOne Strep-coated Magnetic beads per eachcondition into a 1.5 ml eppendorf tube and place on a magnetic beadrack. Have a Bead only control as well (n=3)

19. Remove supernatant and wash 1×500 μl with Bead buffer.

20. Discard supernatant

21. Resuspend beads in an equal volume of 1×PBS, 3 mM MgCl₂ (equal volto what was taken out originally=10 μl)

22. Add the 10 μl of beads directly to the 4780 μL from step 19. To Beadonly control add PBS.

23. Incubate samples with streptavidin beads 1 hr RT on plate shaker(taped).

24. Place on the large magnetic stand for 1 min and remove supernatant

25. Add 1.5 mL of 1× lysis buffer to the samples (do 3×500 μl with agood rinse of the 50 mL falcon tube for each to collect all the beads)and transfer to a new set of eppendorf tubes.

26. Incubate for 20 min on ice.

27. Place tubes in magnetic bead rack, let equilibrate 1 min and removethe supernatant.

28. Wash the beads with wash buffer #1 via vortexing. Resuspend well.

29. Place tubes on magnetic bead rack, let equilibrate 1 min and removethe supernatant

30. Wash 2 additional times as with wash buffer #1 steps 27-29 (total 3washes with wash buffer #1)

31. Repeat steps 27-29 (2) additional times with wash buffer #2

32. During the last wash transfer beads to a new eppendorf tube. (toreduce non-specific binding)

33. Do one dry spin to make sure all residual wash buffer is removed.

34. Add 10 μl of Biotin Elution buffer 1 to beads

35. Incubate for 15 minutes at 37° C.

36. Place on magnetic stand for 1 min, collect sup and transfer to a newtube, add 10 μL of 2× LDS, 2× Reducing agent to eluted sample. Save asElution #1.

37. Add 10 μl of 1×LDS Sample Buffer, 1× Reducing buffer to magneticbeads.

38. Boil the samples for 15 min at 90° C. The boiling time is 15 minutesto essure the streptavidin on the beads unfolds and releases thebiotinylated aptamer-protein complex.

39. Place samples on magnetic stand on ice and collect the elutedsample. This is Elution #2. Discard the beads.

40. Gel 1 layout:

Lane 1: 5 ng Desthiobiotin library

Lane 2: 1×LDS

Lane 3: Marker

Lane 4: Desthiobiotin Elution #1

Lane 5: Unlabeled Elution #1

Lane 6: Bead only Elution #1

Lane 7: Desthiobiotin Elution #2

Lane 8: Unlabeled Elution #2

Lane 9: Bead only Elution #2

Lane 10: Input for AP (saved from step 17)

Running Reducing SDS Gel:

Prepare 1×MOPS SDS Running Buffer from 20×MOPS SDS Buffer

Use 10 or 12 well 4-12% Bis Tris gel

Peel off tape seal and place in the gel box. Insert spacer for secondgel cassette if needed

Fill the inside/upper chamber with running buffer MOPS (1×) and 500 ulAntioxidant

Remove the comb carefully, not disturbing the wells

Rinse the wells with the running buffer to remove the storage bufferwhich can interfere with sample running

Slowly load samples to each well carefully using L-20 tip

Fill the outer/lower chamber with approximately 600 ml of running bufferMOPS (1×)

Place top portion of unit and secure correct electrodes

Run the gel to migrate proteins

100 V constant for samples to move through stack (until all samples lineup) for 15 min

Increase to 150 V constant for running (until visible sample buffercomes to bottom) for ˜1 hr

At the end of the run, stop the power supply and remove the gelcassettes from cell

Disassemble the gel cassette by with gel knife.

Remove one side of cassette case. Trim off the gel foot and wells (avoiddrying gel).

Transfer gel into container filled with Milli Q water and perform aquick wash.

Silver Staining:

Materials:

ProteoSilver TMSilver Stain Kit, Sigma Catalog No. PROT-SIL 1, Lot No.SLBJ0252V

Ethanol, Fisher Scientific Catalog No. BP2818-4, Lot No. 142224

Acetic acid, Acros organics Catalog No. 14893-0025, Lot No. B0520036

Water, Sigma Catalog No. W4502, Lot No. RNBD1581

Preparation:

1. Fixing solution. Add 50 ml of ethanol and 10 ml of acetic acid to 40ml of ultrapure water.

2. 30% Ethanol solution. Add 30 ml of ethanol to 70 ml of ultrapurewater.

3. Sensitizer solution. Add 1 ml of ProteoSilver Sensitizer to 99 ml ofultrapure water. The prepared solution should be used within 2 hours. Aprecipitate may form in the ProteoSilver Sensitizer. This precipitatewill not affect the performance of the solution. Simply allow theprecipitate to settle and remove 1 ml of the supernatant.

4. Silver solution. Add 1 ml of ProteoSilver Silver Solution to 99 ml ofultrapure water. The prepared solution should be used within 2 hours.

5. Developer solution. Add 5 ml ProteoSilver Developer 1 and 0.1 mlProteoSilver Developer 2 to 95 ml of ultrapure water. The developersolution should be prepared immediately (<20 minutes) before use.

6. All steps should be carried out in the hood and waste needs to becollected in toxic designated container.

Procedure

A. Direct Silver Staining

All steps are carried out at room temperature on an orbital shaker at 60to 70 rpm.

1. Fixing—After electrophoresis of the proteins in the minipolyacrylamide gel, place the gel into a clean tray with 100 ml of theFixing solution overnight in the hood. Cover tightly.

2. Ethanol wash—Decant the Fixing solution and wash the gel for 10minutes with 100 ml of the 30% Ethanol solution.

3. Water wash—Decant the 30% Ethanol solution and wash the gel for 10minutes with 200 ml of ultrapure water.

4. Sensitization—Decant the water and incubate the gel for 10 minuteswith 100 ml of the Sensitizer solution.

5. Water wash—Decant the Sensitizer solution and wash the gel twice,each time for 10 minutes with 200 ml of ultrapure water.

7. Silver equilibration—Decant the water and equilibrate the gel for 10minutes with 100 ml of the Silver solution.

8. Water wash—Decant the Silver solution and wash the gel for 1 to 1.5minutes with 200 ml of ultrapure water.

9. Gel development—Decant the water and develop the gel with 100 ml ofthe Developer solution. Development times of 3 to 7 minutes aresufficient to produce the desired staining intensity for most gels.Development times as long as 10 to 12 minutes may be required to detectbands or spots with very low protein concentrations (0.1 ng/mm2)

10. Stop—Add 5 ml of the ProteoSilver Stop Solution to the developersolution to stop the developing reaction and incubate for 5 minutes.Bubbles of CO₂ gas will form in the mixture.

11. Storage—Decant the Developer/Stop solution and wash the gel for 15minutes with 200 ml of ultrapure water. Store the gel in fresh,ultrapure water and take picture for documentation.

Protein Identification

Protein bands of interest were excised from the gradient gels andsubjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS)as above.

Example 12 Use of an Oligonucleotide Probe Library to CharacterizeBreast Cancer Samples

An oligonucleotide probe library comprising approximately 2000 differentprobe sequences was constructed and used to probe approximately 500individual breast cancer and non-cancer samples. The probe sequenceswere derived from different screening experiments and are listed hereinin SEQ ID NOs 10-2921. The oligonucleotides listed in these tables weresynthesized and pooled together. The samples were plasma samples from212 breast cancer patients, 177 biospy confirmed non-cancer patients,and 117 normal control patients (self-reported as non-cancer). Theplasma samples were contacted with the oligonucleotide probe library andmicrovesicles were isolated using PEG precipitation. Oligonucleotidesthat were recovered with the microvesicles were isolated. NextGeneration Sequencing (Illumina HiSeq) was used to identify the isolatedsequences for each sample.

Analysis of significance of difference identified 18 aptamers withp-values below 0.01 when compared Cancer/Normal, 15 aptamers withp-values below 0.001 when compared cancer/Non-Cancer, 28 aptamers withp-values below 0.001 when compared Non-Cancer/Normal.

Multi-oligonucleotide panels were next contructed using across-validation approach. Briefly, 50 samples were randomly withheldfrom the sample cohort. The performance of individual oligonucleotidesto distinguish the remaining cancers and non-cancer/normals wasdetermined using logistic regression methodology. Additionaloligonucleotides were added iteratively and performance was assessedusing logistic regression until further performance improvements were nolonger obtained with additional oligonucleotides. The approach generallyled to panels of approximately 20-100 different probe sequences. Thecontructed panels were then used to classify the 50 withheld samples anddiagnostic performance was assessed using Receiver Operating Curve (ROC)analysis and estimation of the Area under the Curve (AUC).

In approximately 300 rounds of cross-validation, the average AUC was0.6, thus showing that the average performance was statistically betterthan random (i.e., AUC of 0.5) and that the probe library coulddistinguish breast cancer and non-breast cancer/normal patient samples.AUC values as high as 0.8 were observed for particular crossvalidations. FIGS. 7A-B illustrate a model generated using a training(FIG. 7A) and test (FIG. 7B) set from a round of cross validation. TheAUC was 0.803. The variable regions of the sequences used to build thismodel are shown in Table 18. Another exemplary round of cross-validationis shown in FIGS. 7C-D. The AUC was 0.678.

The SEQ ID NOs. of the sequences used in the model in FIGS. 7A-B arelisted in rank in Table 18. The oligonucleotides were synthesized with a5′ region consisting of the sequence (5′-CTAGCATGACTGCAGTACGT (SEQ IDNO. 4)) and a 3′ region consisting of the sequence(5′-CTGTCTCTTATACACATCTGACGCTGCCGACGA (SEQ ID NO. 5)) flanking thevariable regions.

TABLE 18 Oligonucleotide Probe Variable Regions Rank Ordered SEQ ID NOs88, 1057, 834, 1608, 653, 1090, 2803, 499, 2587, 1082, 237, 2873, 2886,759, 287, 390, 472, 119, 289, 96, 380, 459, 1226, 1331, 1012, 2542,1284, 2765, 2528, 334, 1688, 949, 172, 1180, 832, 658, 195, 509, 1015,538, 465, 696, 41, 954, 2771, 55, 407, 1351, 2524, 2760, 1728, 2600,1731, 729, 2920, 156, 1322, 1745, 478, 236, 139, 2911, 2013, 1077, 525,507, 2534, 1041, 1499, 766, 1037, 1143, 912, 1502, 968, 1420

The data presented in this Example demonstrate that an oligonucleotidepool comprising members having the variable regions listed in SEQ ID NOs10-2921, e.g., a pool of probes having the variable regions listed inTable 18, can be used to distinguish plasma from individuals havingbreast cancer versus plasma from non-breast cancer individuals.

Example 13 Single Stranded DNA (ssDNA) Oligonucleotide LibraryPreparation for Library Development

The preparation of high yield and high quality ssDNA libraries is acritical step in SELEX (Systematic Evolution of Ligands by EXponentialenrichment) [1, 2] as well as in other biological applications, such asDNA chips and microarrays [3], and single-stranded conformationpolymorphism technique (SSCP) [4]. The standard approach for preparingssDNA libraries includes PCR amplification to first generate a doublestranded (dsDNA) library, followed by ssDNA separation and purification.Several strategies of ssDNA preparation have been developed to date,each with advantages and disadvantages:

Lambda Exonuclease Digestion [2, 5-7]

The dsDNA standard PCR product is followed by Lambda exonuclease todigest the complementary strand and leave the target ssDNA. ssDNApurification is then performed to remove enzymes and unwanted buffer.

Advantages: Regular PCR amplification has high yield in generatingdsDNA.

Disadvantages: The purity of final ssDNA is limited by enzyme digestionefficiency. Also dsDNA needs to be purified prior to digestion, togetherwith post-digestion purification there will be two purifications, whichresults in substantial loss of input material. The digestion usuallyrequires at least 2 hours. The digestion rate may not be consistent.

Asymmetric PCR [8, 9]

The procedure generates target ssDNA as the main product and less dsDNAproducts and non-target ssDNA. The band corresponding to the targetssDNA is cut from a native gel.

Advantages: The final ssDNA product potentially has high purity.

Disadvantages: Separation of strands is possible in the native gel, butthe yield is typically low and the presence of non-target strand cannotbe excluded. The yield cannot be increased on denaturing gel because thestrands have the same length.

Biotin-Streptavidin Magnetic Beads Separation [10, 11]

The non-target PCR primer is biotinylated so final PCR products areBiotinylated-dsDNA, which can be captured by streptavidin magnetic beadsand denatured to release the non-biotin labeled target ssDNA.

Advantages: The final ssDNA product has relatively high purity.

Disadvantages: In most cases, the input library needs to bebiotinylated, but it may be difficult to replace or release the capturedtarget strands from streptavidin beads. Post-denaturing purification isrequired to remove NaOH and/or acid used for neutralization.

Unequal Primer Length PCR [12]

The non-target PCR primer has a chemical modified spacer and a few extranucleotides following. In the PCR reaction, the DNA polymerase will stopat the spacer, resulting in unequal length of PCR dsDNA product. Thentarget ssDNA can be cut from a denaturing PAGE gel.

Advantages: The final ssDNA product has high purity because the targetssDNA is not mixed with non-target strands.

Disadvantages: ssDNA cannot be seen on native gel. Requires timeconsuming denaturing PAGE gel. It may be difficult to denature somedsDNA library, which can limit the final yield.

Indirect Purification Method [13]

The indirect purification strategy combines Asymmetric PCR andBiotin-streptavidin magnetic beads separation. In short, regular PCR isused to generate sufficient template, then asymmetric PCR with excess oftarget primer and less biotinylated complementary primers, followed bybiotin-streptavidin separation.

Advantages: May increase yield and purity of ssDNA product.

Disadvantages: It cannot produce biotinylated target ssDNA library. Theprocess is relatively long and complicated and may be prone to generatemutants of the original sequence.

The invention provides methods of enriching oligonucleotide probelibraries against a target of interest. As the probes comprise ssDNA,the process may comprise PCR amplification then conversion back intossDNA after each round of enrichment. In this Example, we developed astrategy for preparation of a ssDNA oligonucleotide library. The goalswere to develop a process that is efficient and quick, while deliveringhigh quality/purity ssDNA. We aimed to combine PCR and ssDNA prep in onestep, remain efficient in the presence of selection buffer, targetmolecules, other sample components (e.g., highly abundant proteins forplasma samples) and other assay components (e.g., PEG precipitationsolution that may be used to precipitate microvesicles). In addition, wedesired the method to be able to generate ssDNA library with anymodification, including without limitation Biotin.

We have used an optimized version of Lambda exonuclease digestionprotocol for preparation of ssDNA oligonucleotide library. However, thedigestion yield limits the overall recovery and is not consistentbetween different library preparations. In some cases, the ssDNA band ishardly visible on the gel following digestion. We have also observedincomplete digestion of dsDNA in the ssDNA product. In this Example, wedeveloped an alternative protocol, termed “ssDNA by Unequal lengthPRimer Asymmetric PCR,” or SUPRA. It lacks disadvantages from the knownmethods listed above, and provides high quality and yield up to 10×higher yield of ssDNA oligonucleotide library as compared to theprevious methods. It is relatively fast and convenient technically,since target ssDNA can be distinguished from non-target DNA on a gel.

A schematic comparing standard PCR 900 and unequal length PCR 910 isshown in FIG. 9A. In regular PCR 900, a forward primer 901 and reverseprimer 903 are hybridized with the reverse strand of an aptamer library902. The PCR reaction is performed, thereby creating equal lengthforward 904 and reverse strands 902. The strands are denatured in equallength single strands 905. In unequal length PCR 901, a formard primer911 having a lengthener segment and terminator segment and a reverseprimer 913 are hybridized with the reverse strand of an aptamer library912. The PCR reaction is performed, thereby creating unequal lengthforward 914 and reverse strands 912. The strands are denatured intounequal length single strands 914 and 912 that can be separated by size,e.g., on a denaturing gel.

The steps of SUPRA include: (i) Modification of regular non-targetprimer with two Isp9 (Internal Spacer 9; triethylene glycol spacer) asterminator and 32 extra nucleotides (e.g., poly-A) as lengthener. It isreferred as Unequal-Forward-Double isp9 primer (UF-D9); (ii) Performasymmetric PCR, by mixing DNA template, UF-D9 and regular target(reverse) primer at ratio that favors the reverse primer, e.g., 1:37.5.The PCR program has longer elongation step (e.g., 3 min instead ofstandard 1 min) and more cycles due to linear amplification mode(instead of exponential). The PCR product contains a majority of targetssDNA and small portion of dsDNA. (iii) Mix PCR reaction products 1:1with denaturing buffer (e.g., 180 mM NaOH and 6 mM EDTA) and denaturesamples by heating (e.g., 70° C. for 10 min) and cooling (e.g.,incubation on ice for 3 min); (iv) Run denatured products in denaturingbuffer on an agarose gel stained with SybrGold. The non-target strand,which is longer due to the lengthener, will appear as upper band (ifvisible) and the target strand (strong lower band) is cut and purified.The process can include optional steps, including without limitation:(v) Weigh the gel pieces and purify ssDNA from the gel pieces (e.g.,using the ssDNA Nucleospin kit or the like); (vi) quantification of theyield and native gel can be used to check the purity and yield of finalproduct (e.g., using the ssDNA Qubit kit or the like).

The first step (i) uses a specific design of the forward primer withefficient terminator and lengthener, which creates non-target strand ofunequal length. The DNA polymerase used to build the target strand willstop polymerization once it reaches the terminator, and the lengthenerfacilitates differentiation between the target and non-target strands.In the second step (ii), the ratio between the two primers is shiftedtoward the reverse primer, to produce a majority of target ssDNA. Theratio, however, should not limit double strand templates production tokeep reaction running FIG. 9B is a gel showing titration of forward andreverse primers input in asymmetrical PCR. The optimal condition, atwhich target strand is clearly visible, is in the range 1:20-1:50 F:Rprimers ratio. As shown in the figure, the ratio between two primers inasymmetric PCR can affect dsDNA and ssDNA amount in final products. ThePCR thermocycler program is also adjusted to provide efficiency in theasymmetric PCR. In the third step (iii), a reliable denaturing method isused to separate target ssDNA to ensure the final yield and high purity.

As desired, the final step (vi) estimates the ratio of residual dsDNA,e.g., using ssDNA Qubit kit. In cases where the yield is not critical,the denaturing steps (iii and iv) can be skipped and the PCR productscan be directly run on native gel. There will be a dsDNA band, but lowerMW target ssDNA band can be distinguished and purified from gel. This isalso a way to visualize the target band directly after PCR for a qualitycheck or purification without denaturing. The purity of final productwill be the same but yield will be lower.

A comparison of native versus denatured gel purification is shown inFIG. 9C. A post-probing oligonucleotide probe library was PCRed usingunequal length primers mixed at a ratio of 1:38 (Forward/Reverse). Inthe figure, the left lane on each gel is a 50 bp molecular weight ladderand the lower band is the reverse primer. The positions of the dsDNA andss DNA are indicated. A native gel showed the presence of both dsDNA andssDNA (target strand) (FIG. 9C, panel A). Here, part of the targetreverse strand is migrating in dsDNA. Thus, using the native gel, onecan purify target ssDNA with moderate recovery. When a higher yield isdesired, the PCR products can be run on denaturing agarose gel asdescribed above. This approach provides maximal recovery wherein onlytarget strand is visible, and can be cut from gel and purified (FIG. 9C,panel B). In this case, the reverse strand ssDNA, which is part of thedsDNA on native gel (FIG. 9C, panel A), is denatured and migratestogether with other free molecules of target ssDNA strand, while forwardstrand becomes invisible due to limited amplification.

Compared to standard asymmetric PCR, which has relatively low yield anddoes not allow to distinguish target and non-target strands ondenaturing gel, SUPRA delivers different lengths of target andnon-target that can be purified on both native gel and denaturing gels.Compared to unequal primer length PCR, which uses lengthy Urea-PAGEprotocol and produces only dsDNA, SUPRA has less dsDNA and free targetssDNA can be cut even from native gel if yield is not critical.

SUPRA has been used in the oligonucleotide probe library enrichmentmethods provided by the invention. The method is robust. In the presenceof enrichment buffer, target/non-target molecules, proteins,exosomes/microvesicles, PEG and other components, SUPRA provides highquality and quantity of the ssDNA oligonucleotide library.

References

-   1. Comparison of different methods for generation of single-stranded    DNA for SELEX processes. Anal. Bioanal. Chem. 2012, 404, 835-842.-   2. Upgrading SELEX Technology by Using Lambda Exonuclease Digestion    for Single-Stranded DNA Generation. Molecules 2010, 15, 1-11.-   3. Tang, K.; Fu, D. J.; Julien, D.; Braun, A.; Cantor, C. R.;    Koster, H. Chip-based genotyping by mass spectrometry. Proc. Natl.    Acad. Sci. USA 1999, 96, 10016-10020.-   4. Kuypers, A. W.; Linssen, P. C.; Willems, P. M.; Mensink, E. J.    On-line melting of double-stranded DNA for analysis of    single-stranded DNA using capillary electrophoresis. J. Chromatogr.    B Biomed. Appl. 1996, 675, 205-211.-   5. Higuchi, R. G.; Ochman, H. Production of single-stranded DNA    templates by exonuclease digestion following the polymerase chain    reaction. Nucleic Acids Res. 1989, 17, 5865.-   6. Jones, L. A.; Clancy, L. E.; Rawlinson, W. D.; White, P. A.    High-affinity aptamers to subtype 3a hepatitis C virus polymerase    display genotypic specificity. Antimicrob. Agents Chemother. 2006,    50, 3019-3027.-   7. S. S. Oh, K. Ahmads, M. Cho, Y. Xiao, H. T. Soh, “Rapid,    Efficient Aptamer Generation: Kinetic-Challenge Microfluidic SELEX,”    presented in the 12th Annual UC Systemwide Bioengineering Symposium,    June 1315, 2011, Santa Barbara, U.S.A-   8. Gyllensten, U. B.; Erlich, H. A. Generation of single-stranded    DNA by the polymerase chain reaction and its application to direct    sequencing of the HLA-DQA locus. Proc. Natl. Acad. Sci. USA 1988,    85, 7652-7656.-   9. Wu, L.; Curran, J. F. An allosteric synthetic DNA. Nucleic Acids    Res. 1999, 27, 1512-1516.-   10. Espelund, M.; Stacy, R. A.; Jakobsen, K. S. A simple method for    generating single-stranded DNA probes labeled to high activities.    Nucleic Acids Res. 1990, 18, 6157-6158.-   11. A. Paul, M. Avci-Adali, G. Ziemer, H. P. Wendel.    Streptavidin-coated magnetic beads for DNA strand separation    implicate a multitude of problems during cell-SELEX.    Oligonucleotides 2009, 19, 243-254.-   12. Williams K., Bartel D. PCR product with strands of unequal    length. Nucleic Acids Research, 1995, Vol. 23, No. 20.-   13. Indirect purification method provides high yield and quality    ssDNA sublibrary for potential aptamer selection. Anal. Biochem.    2015, online available.

Example 14 Oligonucleotide Pools to Characterize Cell Lines

In this Example, an oligonucleotide library was enriched using acombination of cells from different cancer cell lines to create cancerspecific pools and against a pool of non-cancer cells. The enrichment isperformed to identify cancer specific oligonucleotides andoligonucleotide pools that can be used in various applications,including without limitation diagnostic assays, as drugs or in drugdelivery.

The unscreened library comprised F-Trin-B primers (i.e., 5′CTAGCATGACTGCAGTACGT (SEQ ID NO 4) and 5′CTGTCTCTTATACACATCTGACGCTGCCGACGA (SEQ ID NO 5) as shown above)surrounding a region of randomly generated nucleotides. The enrichmentwas performed using methodology presented herein. See Examples above.Detailed protocols are below in Examples 15 and 16. One round ofenrichment consisted of a series of positive, negative and positiveselections before amplification via PCR of the enrichedoligonucleotides. See FIG. 13, which shows a diagram of one round ofenrichment. The amplified library (PCR) is used as the input into thenext round or enrichment. The cell lines used consisted of nine lungcancer lines, five prostate cancer lines and nine non-cancer lines aslisted in Table 19.

TABLE 19 Cell Lines Indication Cell Line Tissue Disease Morphology LungPool A549 Lung Carcinoma Epithelial NCI-H1395 Lung AdenocarcinomaEpithelial NCI-H1838 Lung Adenocarcinoma; Non-Small Epithelial Cell LungCancer NCI-H1975 Lung Adenocarcinoma; Non-Small Epithelial Cell LungCancer NCI-H2122 Lung; Derived From Adenocarcinoma; Non-Small RoundedMetastatic Site: Pleural Cell Lung Cancer And Effusion Epithelial CellsNCI-H460 Lung; Pleural Effusion Carcinoma; Large Cell Lung EpithelialCancer H69AR Lung Carcinoma; Small Cell Lung Epithelial Cancer HCC827Lung Adenocarcinoma Epithelial NCI-H1688 Lung; Derived From Carcinoma;Small Cell Lung Epithelial Metastatic Site: Liver Cancer Prostate 22RV1Prostate Carcinoma Epithelial Pool DU145 Prostate; Derived FromCarcinoma Epithelial Metastatic Site: Brain LnCaP Prostate; Derived FromCarcinoma Epithelial Metastatic Site: Left Supraclavicular Lymph NodePC3 Prostate; Derived From Carcinoma Epithelial Metastatic Site: BoneVCaP Prostate; Derived From Cancer Epithelial Metastatic Site: VertebralMetastasis Non-Cancer CCD-16Lu Lung Normal Fibroblast Pool CCD-19Lu LungNormal Fibroblast CCD841CoN Colon Normal Epithelial CCD-18Co ColonNormal Fibroblast HCC1143BL B Lymphoblast; Peripheral Normal LymphoblastBlood Lymphocytes NCI-BL1395 Peripheral Blood; B Normal LymphoblastLymphoblast; Epstein-Barr Virus (EBV) Transformed NCI-BL128 PeripheralBlood; B Normal Lymphoblast Lymphoblast; Epstein-Barr Virus (EBV)Transformed Primary Prostate Normal Epithelial Prostate Epithelial CellsPNT2 Prostate Normal Epithelial

Nine rounds of enrichment were performed against the lung cancer samplepool. The variable regions of the top five most enriched sequences areshown in Table 20. The enriched libraries consisted of the VariableRegion as shown in the table inserted between the flanking sequencesshown above as 5′ CTAGCATGACTGCAGTACGT (SEQ ID NO 4)—[VariableRegion]—CTGTCTCTTATACACATCTGACGCTGCCGACGA (SEQ ID NO 5). The sequenceswere also 5′ biotinylated. 25 ng of starting library was used in theenrichment. As control sequences, the reverse complements weresynthesized as shown in Table 20. These are reverse complements of theentire oligonucleotides including the flanking regions.S1LCa25-R9S1RC-5′biotin and S1LCa25-R9S3RC-5′biotin are the reversecomplements of S1LCa25-R9S1-5′biotin and S1LCa25-R9S3-5′biotin,respectively. The reverse complements can be used as negative controlsas they should not specifically bind the targets of the enrichedsequences.

TABLE 20 Lung Cancer Enriched Oligonucleotides Sequence SEQ ID name NO.Variable Region (5′->3′) Most S1LCa25-GGGGTTGTTTTGGGATGCCTTTTTCTCTGTATTTCA 2922 enriched R951- sequences5′biotin S1LCa25- GTCCTCGCCCGGGCTTCTGTTTGTTTTTTGGATTCGA 2923 R952-5′biotin S1LCa25- AACGCTTGATTTGGGTGGTTGGATTGACCTTTTTATGA 2924 R953-5′biotin S1LCa25- TTTTTTATTGGGTGCGCATAGGCGAGTGGTCTCTT 2925 R954-5′biotin S1LCa25- TGATTACATCGCCTGTATGGGTTGTTGTTTGTGTC 2926 R955-5′biotin Full Sequence (5′->3′) Reverse S1LCa25-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGAAA 2927 complement R9S1RC-TACAGAGAAAAAGGCATCCCAAAACAACCCCACGTACT sequences 5'biotin GCAGTCATGCTAGS1LCa25- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCATA 2928 R9S3RC-AAAAGGTCAATCCAACCACCCAAATCAAGCGTTACGTA 5′biotin CTGCAGTCATGCTAG

Nine rounds of enrichment were also performed against the prostatecancer sample pool. The variable regions of various enriched sequencesare shown in Table 21. The amount of starting library used in theenrichment is indicated in the table. The libraries consisted of theVariable Region as shown in the table inserted between the flankingsequences (SEQ ID NOs 4-5) as shown above. After the enrichment, thelibraries were used to probe the cell pools and NGS was used todetermine the identity and counts of the bound sequences. Table 21 showsthe counts of sequences in prostate cancer (PCA) or non-PCA and thefold-change between PCA and non-PCA. Probing was performed intriplicate. The sequences in Table 30 had a coefficient of variation (%CV)≤20% across normalized counts for three probing replicates and foldchanges cancer/non-cancer≤0.6 or ≥1.4. The average percent variation (%CV) was ˜11% for the PCA pools and ˜12% for the non-PCA pools.

TABLE 21 Prostate Cancer Enriched Oligonucleotides PCA Non-PCA FoldAverage Average Change - SEQ normalized normalized Cancer/Non-Variable Region (5′->3′) ID NO counts counts Cancer EnrichmentGGTTTTATCGTTTCTTTAGTTGGGTTCTTGGG 2929 475 346 1.4 with 5 ng TGAinitially GGATCTTGGTTAGTATTTTTGGTATTTTCTGT 2930 415 300 1.4 GGTGGATGCTGGTTAGTATTTTTGGTATTTTCTGT 2931 564 408 1.4 GGTTATTTAGGGGTTGTGGGTCTAATTTTTGTTTG 2932 551 397 1.4 TTCGA EnrichmentTCCTGGTTTCTGGTGGTTTCATTTAGCTTGTT 2933 376 276 1.4 with 25 ng ACCTGAinitially TCCTGGTTTCTGGTGGTTTAATTTTGCTTGTT 2934 934 687 1.4 ACATGATCCTGGTTTCTGGTGGTTTCATTTTGATTGTT 2935 2036 1485 1.4 ACCT GATTTGGTTGGTCCATGGGTAAGCTTGGTGATTC 2936 438 315 1.4 TCTTGATCCTGGTTTCTGGTGGTTTCATTTTGCTTGTT 2937 2194 1565 1.4 ACATGA EnrichmentACATGCACTGAGCCCGACACACCCGCCTGAAC 2938 361 570 0.6 with 50 ng TATinitially ACTAATTGTTTTGGGGGTAGTTGTTTTTTTTC 2939 3984 2893 1.4 TGTGGATCCTGGTTAGTATTTTTGGTATATTCTGT 2940 277 201 1.4 GGTTATGTTCTTTTTATTTTAGTGGTTGTGGCCTA 2941 506 367 1.4 TCTA

The sequences above were 5′ biotinylated for capture. As controlsequences, reverse complements were synthesized as shown in Table 22.These are reverse complements of the entire oligonucleotides includingthe flanking regions. S1PCa5-R9S1RC-5′biotin and S1PCa25-R9S1RC-5′biotinare the reverse complements of the complete S1PCa5-R9S1-5′biotin andS1PCa25-R9S1-5′biotin sequences with flanking regions, respectively. Thereverse complements can be used as negative controls as they should notspecifically bind the targets of the enriched sequences.

TABLE 22 Prostate Cancer Enriched Oligonucleotides Sequence nameSEQ ID NO Variable Region (5′->3′) Enrichment S1PCa5-GGTTTTATCGTTTCTTTAGTTGGGTTCTTGGGTGA 2942 with 5 ng R951-5′biotininitially S1PCa5- TATTTAGGGGTTGTGGGTCTAATTTTTGTTTGTTC 2943 R954-5′biotinGA Enrichment S1PCa25- TCCTGGTTTCTGGTGGTTTCATTTAGCTTGTTACC 2944with 25 ng R951-5′biotin TGA initially S1PCa25-TCCTGGTTTCTGGTGGTTTCATTTTGCTTGTTACA 2945 TGA Enrichment S1PCa50-ACATGCACTGAGCCCGACACACCCGCCTGAACTAT 2946 with 50 ng R951-5′biotininitially S1PCa50- GGATCCTGGTTAGTATTTTTGGTATATTCTGTGGT 2947R953-5′biotin Full Sequence Reverse S1PCa5-/5Biosg/TCGTCGGCAGCGTCAGATGTGTATAAG 2948 complement R9S1RC-AGACAGTCACCCAAGAACCCAACTAAAGAAACGAT sequences 5'biotinAAAACCACGTACTGCAGTCATGCTAG to 5 ng library Reverse S1PCa25-/5Biosg/TCGTCGGCAGCGTCAGATGTGTATAAG 2949 complement R9S1RC-AGACAGTCAGGTAACAAGCTAAATGAAACCACCAG sequences 5′biotinAAACCAGGAACGTACTGCAGTCATGCTAG to 25 ng library

Additional sequences as in Table 21 are shown in Table 23. Table 23includes sequences with % CV>20%, as indicated.

TABLE 23 Additional Prostate Cancer Enriched Oligonucleotides ProstateNon-Cancer Cancer Pool Pool Fold Average Average Change - SEQ IDnormalized % normalized % Cancer/Non- Variable Region (5′->3′) NO countsCV counts CV Cancer Enrichment AGTTCTTGGGGGTTTTGGTTGG 2950 670 10% 26641% 2.5 with 5 ng TGCCTTGTATGTTA initially AGTTCTTGGGGGTTTTGGTTGT 295111 60% 1  0% 10.7 TGCCTTGTCTATTA Enrichment TTGCCGCCCTTTATGGTTTGTT 295245 49% 8 90% 5.4 with 25 ng TTTTGCGATGTGGGA initially

Assays such as qPCR, cell enzyme linked assay (ELA), confocal microscopyand cell viability assays are performed to verify binding ofoligonucleotides to cells and identify potential cell killing propertiesof certain oligonucleotides.

Example 15 Enrichment Protocol

Re-amplification of original F-TRin-35n-B oligonucleotide library.

Samples for One Round of Enrichment

900,000 cells from pool of Prostate/Lung cell lines (split 3 times in300,000 each)

900,000 cells from pool of Normal cell lines (split 3 times in 300,000each)

Preparations

Pre-chill tabletop centrifuge at 4° C.

Bring 300,000 of Prostate/Lung cells to 140 ul with 1×PBS with 3 mMMgCl₂ (138.6 ul PBS+1.4 ul 300 mM MgCl₂), add competitors listed belowand incubate for 20 min with end-over-end rotation.

Mixture of two competitors: Salmon DNA+tRNA.

Competitor salmon DNA: add 800 ng of salmon DNA (Stock 10 ug/ul→Dilute1:250 with 1×PBS+3 mM MgCl₂ [40 ng/ul]→20 ul input).

Competitor tRNA: add 800 ng of tRNA (Stock 10 ug/ul→Dilute 1:250 with1×PBS+3 mM MgCl₂ [40 ng/ul]→20 ul input).

Library/sample incubation (positive selection)

Prepare 5, 25, 50 ng for first round and same corresponding forfollowing rounds of oligonucleotide library in 20 ul of 1×PBS with 3 mMMgCl₂. After heating the DNA for 3 min@95 C, put it on ice immediatelyfor 5 min. Add DNA to the blocked cells and incubate them for 30 min atRT, with end-over-end rotation

Spinning Cells

Spin cells from step II at 500×g for 5 min (spin at 4 C) and discardsupernatant by pipetting it out.

Re-suspend pellet in 1 ml of 1×PBS+3 mM MgCl₂; vortex/mix and centrifugeagain at 500×g for 5 min (spin at 4 C)

Repeat the wash one more time.

Re-suspend pellet in 50 ul of water.

Use pellet in step IV.

Oligonucleotide Elution

Add 25 ul of 0.1N NaOH to cells from III, incubate for 10 min at 50 C,mixmate and agitate for 10 sec at 550 rpm→Add 25 ul of 0.1N HCL→Spin at12000×g for 10 min at 4 C→proceed with NucleoSpin ssDNA purificationMAKE SURE TO USE CORRECT BUFFER TO BIND (NTC) ssDNA (number of columnsto be identified)→Elute, each column in 20 ul of water (incubation timebefore elution is 5 min). Add 4 ul of 5×PBS+15 mM MgCl2 solution, beforeproceeding to the next enrichment.

Library/Sample Incubation (Negative Selection)

Bring 300,000 of Normal cells (depending on number of steps) to 140 ulwith 1×PBS with 3 mM MgCl₂ (138.6 ul PBS+1.4 ul 300 mM MgCl₂) and mixwith the mixture of competitors as described above in step I.c (40 ultotal), incubate for 20 min with end-over-end rotation. Then, add elutedoligonucleotide libraries (˜20 ul) (heat treat 3 min@95 C, put it on iceimmediately for 5 min) from step IV and incubate 30 min at RT.

Spinning Cells

Spin cells from step V at 500×g for 5 min (spin at 4 C) and collectsupernatant.

Treatment of supernatant with NucleoSpin ssDNA purification. MAKE SURETO USE CORRECT BUFFER TO BIND (NTC) ssDNA (number of columns to beidentified)→Elute, each column in 20 ul of water (incubation time beforeelution is 5 min).

Add 4 ul of 5×PBS+15 mM MgCl2 solution, before proceeding to the nextenrichment.

Library/Sample Incubation (Positive Selection)

Bring 300,000 of Prostate/Lung cells to 140 ul with 1×PBS with 3 mMMgCl₂ (138.6 ul PBS+1.4 ul 300 mM MgCl₂) and mix with the mixture ofcompetitors as described above in step I.c (40 ul total), incubate for20 min with end-over-end rotation. Mix with libraries from step VI (heattreat 3 min@95 C, put it on ice immediately for 5 min) and incubate for30 min at RT with end-over-end rotation.

Spinning Cells

Spin cells from step VII at 500×g for 5 min (spin at 4 C) and discardsupernatant by pipetting it out.

Re-suspend pellet in 1 ml of 1×PBS+3 mM MgCl₂; vortex/mix and centrifugeagain at 500×g for 5 min (spin at 4 C).

Repeat the wash one more time.

Re-suspend pellet in 50 ul of water.

Follow the oligonucleotide elution (IV) and then nucleospin purificationto get the ssDNA in 20 ul water.

The ssDNA in water (20 ul) will be used entirely for PCR amplification.

Example 16 Enrichment Protocol

Samples for one round of enrichment

900,000 cells from pool of Prostate/Lung cell lines (split 3 times in300,000 each)

900,000 cells from pool of Normal cell lines (split 3 times in 300,000each)

No cells enrichment for every library input

TABLE 24 Buffers Exp. ID Buffers B1 PBS + 3 mM MgCl2 B2 PBS + 3 mMMgCl2 + 0.5% F-127 + 0.5% PEG4000 B3 PBS + 3 mM MgCl2 + 0.5% F-127 + 1mg/ml HSA

Or the titers of buffer with F-127 and H SA-F-127@0.5, 1 and 2%

Cells are available in 1×PBS+3 mM MgCl2 buffer and need to betransferred.

I. Preparations

Pre-chill tabletop centrifuge at 4° C.

Transfer 300,000 cancer cells—140 ul into 3 fresh tubes. Spin at 500×gfor 5 mins. Remove the supernatant buffer and add 70 ul of the bufferB2.

Add competitors listed below and incubate for 20 min with end-over-endrotation.

Mixture of two competitors:

-   -   a. Competitor salmon DNA: add 800 ng of salmon DNA (Stock 10        ug/ul→Dilute 1:125 with B2 [80 ng/ul]→10 ul input). b.        Competitor t-RNA: add 800 ng of tRNA (Stock 10 ug/ul→Dilute        1:125 with B2 [80 ng/ul]→10 ul input).

Bring up the volume to 180 ul with 90 ul of Buffer B2, for the blocking.

II. Library/Sample Incubation (Positive Selection)

Prepare 5/25/50 ng of F-TRin-35n-B Starting Library for first round in20 ul of buffer B2. After heating the DNA for 3 min@95 C, put it on iceimmediately for 5 min. Add DNA to the blocked cells and incubate themfor 30 min at RT, with end-over-end rotation.

III. Spinning Cells

Spin cells from step II at 500×g for 5 min at 4 C and discardsupernatant by pipetting it out.

Re-suspend pellet in 1 ml of B2; vortex and centrifuge again at 500×gfor 5 min at 4 C.

Repeat the wash one more time. (Additional Wash)

Re-suspend pellet in 30 ul of buffer B2.

Use pellet in step IV.

IV. Oligonucleotide Elution

Add 10 ul of 0.25N NaOH to cells from III, incubate for 10 min at 50 C,mix-mate and agitate for 5-10 sec at 550 rpm→Add 10 ul of 0.25N HCL→Spinat 16000×g for 10 min at 4 C. Collect the supernatant; this will be usedas library for the next step.

V. Library/Sample Incubation (Negative Selection)

Transfer 300,000 normal cells—140 ul into 3 fresh tubes. Spin@500×g for5 mins. Remove the supernatant buffer and add 70 ul of the buffer B2.

Mix with the mixture of competitors (10 ul of Salmon sperm DNA@80ng/ul+10 ul of Yeast t-RNA@80 ng/ul), bring up the volume to 150 ul (add60 ul) of Buffer B2, incubate for 20 min with end-over-end rotation.Then, add eluted oligonucleotide libraries (˜50 ul) (heat treat for 3min@95 C, put it on ice immediately for 5 min) from step IV and incubate30 min at RT.

VI. Spinning Cells

Spin cells from step V at 500×g for 5 min at 4 C and collect supernatantas library for next step (200 ul).

VII. Library/Sample Incubation (Positive Selection)

Transfer 300,000 cancer cells—140 ul into 3 fresh tubes. Spin at 500×gfor 5 mins. Remove the supernatant buffer and add 70 ul of the bufferB2.

Mix with the mixture of competitors (10 ul of Salmon sperm DNA@80ng/ul+10 ul of Yeast t-RNA@80 ng/ul), incubate for 20 min withend-over-end rotation. Mix with libraries from step VI (heat treat 3min@95 C, put it on ice immediately for 5 min) and incubate for 30 minat RT with end-over-end rotation (total 290 ul).

VIII. Spinning Cells

Spin cells from step VII at 500×g for 5 min at 4 C and discardsupernatant by pipetting it out.

Re-suspend pellet in 1 ml of B2; vortex and centrifuge again at 500×gfor 5 min at 4 C.

Repeat the wash one more time. (Additional Wash)

Re-suspend pellet in 50 ul of water; it will be used entirely for PCRamplification.

Example 17 Oligonucleotide Pools to Characterize Cell Line Microvesicles

In this Example, an oligonucleotide library was enriched usingmicrovesicles shed from various cancer cell lines. The enrichment isperformed as in Example 14 as above except that the sample comprisedmicrovesicles as opposed to cells. This method can be performed toidentify disease specific oligonucleotides and oligonucleotide poolsthat can be used in various applications, including without limitationdiagnostic assays, as drugs or in drug delivery.

The unscreened library comprised F-Trin-B primers (i.e., 5′CTAGCATGACTGCAGTACGT (SEQ ID NO 4) and 5′CTGTCTCTTATACACATCTGACGCTGCCGACGA (SEQ ID NO 5)) flanking a randomvariable region. Enrichment was performed on microvesicles from celllines as described further in Example 18 below. FIG. 14A shows copiesper species of five rounds of enrichment on exosomes from VCaP and LNCaPcells as indicated in the figure. Positive selection was performed onVCAP microvesicles shed from VCAP cells and negative selection wasperformed on LNCaP microvesicles shed from LNCaP cells. The figure showsthat the copies numbers of various oligonucleotide probe speciesincreased with each round, indicating that the oligonucleotide probelibrary is being enriched with those species. The variable regions ofseveral enriched oligonucleotide probes are shown in Table 25. Theprobes are 5′ biotinylated. These probes are from the enrichmentperformed with 5 ng library on exosomes from VCaP and LnCaP cells. Table25 shows nine sequences with fold changes of at least 4.0-fold elevatedin VCaP versus LNCaP. Each sequence also had averaged normalized countsof at least 500 for probing on VCaP exosomes. Fold changes are shown inthe table. FIG. 14B shows copies recovered after probing (see Examplebelow for protocol) with the nine oligonucleotide probes in the table.Also as shown in the table, the reverse complement of the entire probesequences (i.e., including the F-Trin-B primers shown above) are used ascontrols.

TABLE 25 Variable Regions of Oligonucleotide probes Fold Change SEQ ID(VCaP / Variable Regions (5′->3′) NO LNCaP)ATATGGGGTTTATGGGGATGGTGTTATGGGTGGAATGA 2953 5.4ATGGGGAGGGGGGTAGGCTGTCTTAATTGGTGGTT 2954 4.3ATTAATGGGTGGGGGGTTTAGCTTGATGTGGGTTGTGA 2955 4.0GAATGGGGGGATACTGTTAGTGTGGGTCTGGGGGT 2956 6.7GGGGGGGGCTTTTTATGGTTTCTGGGGGACCTGCT 2957 4.9GGTGATGAATTAAATGGGGGGGGTATCAAGTGTGGA 2958 6.5TACTTAATTGGGGGGGGGGATTCTGTTTTGTCTCT 2959 6.2TAGCCTTTGGGGGTTGTTTTGGGGGATTGGGTTGTTGA 2960 6.3TAGTGACTACGGGTATGGGGATTGGGGGTTTGGTTTGA 2961 4.5 SEQ IDReverse Complements (5′->3′) NO/5Biosg/TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCATTCCACCCATAACACCA 2962TCCCCATAAACCCCATATACGTACTGCAGTCATGCTAG/5Biosg/TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAACCACCAATTAAGACAGCC 2963TACCCCCCTCCCCATACGTACTGCAGTCATGCTAG/5Biosg/TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCACAACCCACATCAAGCTA 2964AACCCCCCACCCATTAATACGTACTGCAGTCATGCTAG/5Biosg/TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACCCCCAGACCCACACTAAC 2965AGTATCCCCCCATTCACGTACTGCAGTCATGCTAG/5Biosg/TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGCAGGTCCCCCAGAAACCA 2966TAAAAAGCCCCCCCCACGTACTGCAGTCATGCTAG/5Biosg/TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCCACACTTGATACCCCCCC 2967CATTTAATTCATCACCACGTACTGCAGTCATGCTAG/5Biosg/TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGAGACAAAACAGAATCCCC 2968CCCCCCAATTAAGTAACGTACTGCAGTCATGCTAG/5Biosg/TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCAACAACCCAATCCCCCAA 2969AACAACCCCCAAAGGCTAACGTACTGCAGTCATGCTAG/5Biosg/TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCAAACCAAACCCCCAATCC 2970CCATACCCGTAGTCACTAACGTACTGCAGTCATGCTAG

FIG. 14C shows higher recovery on six out of seven batches of isolatedVCaP exosomes compared to exosomes from LNCaP cells as seen by probingwith a library of the nine individual DNA sequences in Table 25. Thefigure also shows probing with the reverse complement controls. Asexpected, these sequences showed little recovery with the exosomes ascompared to the oligonucleotide probe sequences with variable regionsSEQ ID NOs 2953-2961.

Several proteins have been identified by probing of exosomes from VCaPcells with an aptamer having variable region5′-TACTTAATTGGGGGGGGGGATTCTGTTTTGTCTCT-3′ (i.e., SEQ ID NO 2959)followed by pull downs and analysis by mass spectrometry. See Example 9for methodology. Exemplary results are shown in Table 26. Theseexperiments identified key proteins in the regulation of exosomebiogenesis (ESCRT, Syntenin) and endocytic trafficking (chemokineCXCL11) overexpressed in Cancer. The ESCRT machinery (ESCRT-0, I, II andIII) participates in exosomes biogenesis and the proteins have beenobserved to be overexpressed in human cancers. See, e.g, Kowal et al.,Biogenesis and secrection of exosomes, Current Opinion in Cell Biology,2014, 29:116-125; Hurley and Hanson, Membrane budding and scission bythe ESCRT machinery: it's all in the neck, Nat Rev Mol Cell Biol, 201011:556-566; Raiborg and Stenmark, The ESCRT machinery in endosomalsorting of ubiquitylated membrane proteins, Nature 2009 458: 45-52, allof which proteins are incorporated by reference herein in theirentirety. The experiments further identified cold shock proteins whosemiRNA suppression sensitizes cells to chemotherapeutic agents.

TABLE 26 Proteins from VCaP exosomes pulled down with Sequence 7Accession Description Cellular role O14625 C-X-C motif chemokine 11 GN =CXCL11 chemokine that is overexpressed in blood (alsoInterferon-inducible T-cell and tissue of men with advanced prostatealpha chemoattractant (I-TAC)) adenocarcinomas Q92616 Translationalactivator GCN1 GN = GCN1 Q9H444 Charged multivesicular body protein 4bESCRT-III; membrane scission GN = CHMP4B Q14767 Latent-transforminggrowth factor beta- binding protein 2 GN = LTBP2 P26599-3 Isoform 3 ofPolypyrimidine tract-binding cancer associated splicing factor protein 1GN = PTBP1 (also Heterogeneous nuclear ribonucleoprotein I (hnRNP I))P98179 RNA-binding protein 3 GN = RBM3 part of ESCRT-III; membranescission; cold shock protein. Knock-down has been shown to enhancechemotherapeutic cell killing of prostate cells O43633 Chargedmultivesicular body protein 2a GN = CHMP2A P62888 60S ribosomal proteinL30 GN = RPL30 Q9UK41-2 Isoform 2 of Vacuolar protein sorting- part ofESCRT-I; membrane budding with associated protein 28 homolog GN = VPS28ESCRT-II O60884 DnaJ homolog subfamily A member 2 GN = DNAIA2 Q14011-3Isoform 3 of Cold-inducible RNA-binding cold shock protein. Knock-downhas been protein GN = CIRBP (A18 hnRNP) shown to enhancechemotherapeutic cell killing of prostate cells Q8N684-3 Cleavage andpolyadenylation specificity factor subunit 7 GN = CPSF7 P05109 ProteinS100-A8 GN = S100A8 P05386 60S acidic ribosomal protein P1 GN = RPLP1Q7LBR1 Charged multivesicular body protein 1b associated to ESCRT-III;regulation of GN = CHMP1B membrane scission and ESCRT-III disassemblyO00560-1 Syntenin-1 GN = SDCBP adaptor protein that binds to syndecansand ALIX which interacts with multiple ESCRT proteins

Negative controls from the above enrichment with 5 ng library onexosomes from VCaP and LnCaP cells were also identified. Several areshown in Table 27. The Table shows 3 sequences with fold changes of atleast 4.0 higher on LNCaP exosomes and 2 sequences with fold changes of1 (indicating no fold change between VCaP and LNCaP exosomes). Thevariable regions are shown in Table 27. Also as shown in the table, thereverse complement of the entire probe sequences (i.e., including theF-Trin-B primers shown above) are used as controls.

TABLE 27 Variable Regions of Oligonucleotide probes Fold change SEQ ID(VCaP/ Variable Regions (5′→3′) NO. LNCaP) TCCGTTTATCTACTTTTCCGGTAC 29710.2 TGTTCCCGTTT ATCGCGTCGCCCCCGGATATTATT 2972 0.2 GTTTCTTGTTCTTGCTTGCCCGGCCATAAACACGA 2973 0.2 TCTTGTTCTCTA GATACGGTCTTTGGTGCTTGTGTG2974 1.0 AATCTATGGGGTGA TCCTGGTTTCTGGTGGTTTATTTA 2975 1.0 GCTTGTTACCTGAAGTGGGTGGTGGGTTCGGTTTGCT 2976 TGGTTCCCTGTTGA ATTGAGGTGGTTTTGAGGTGGGCT2977 ATCTGAGGGAT GGGGGGGGCTTTTTATGGTTTCTG 2978 GGGGACCTGCTATATGGGGTTTATGGGGATGGTGT 2979 TATGGGTGGAATGT SEQ IDReverse Complements (5′→3′) NO /5Biosg/TCGTCGGCAGCGTCAGATG 2980TGTATAAGAGACAGAAACGGGAACAGT ACCGGAAAAGTAGATAAACGGAACGTA CTGCAGTCATGCTAG/5Biosg/TCGTCGGCAGCGTCAGATG 2981 TGTATAAGAGACAGGAACAAGAAACAATAATATCCGGGGGCGACGCGATACGTA CTGCAGTCATGCTAG /5Biosg/TCGTCGGCAGCGTCAGATG2982 TGTATAAGAGACAGTAGAGAACAAGAT CGTGTTTATGGCCGGGCAAGCAAACGTACTGCAGTCATGCTAG /5Biosg/TCGTCGGCAGCGTCAGATG 2983TGTATAAGAGACAGTCACCCCATAGAT TCACACAAGCACCAAAGACCGTATCACGTACTGCAGTCATGCTAG /5Biosg/TCGTCGGCAGCGTCAGATG 2984TGTATAAGAGACAGTCAGGTAACAAGC TAAATAAACCACCAGAAACCAGGAACGTACTGCAGTCATGCTAG /5Biosg/TCGTCGGCAGCGTCAGATG 2985TGTATAAGAGACAGTCAACAGGGAACC AAGCAAACCGAACCCACCACCCACTACGTACTGCAGTCATGCTAG /5Biosg/TCGTCGGCAGCGTCAGATG 2986TGTATAAGAGACAGATCCCTCAGATAG CCCACCTCAAAACCACCTCAATACGTA CTGCAGTCATGCTAG/5Biosg/TCGTCGGCAGCGTCAGATG 2987 TGTATAAGAGACAGAGCAGGTCCCCCAGAAACCATAAAAAGCCCCCCCCACGTA CTGCAGTCATGCTAG /5Biosg/TCGTCGGCAGCGTCAGATG2988 TGTATAAGAGACAGACATTCCACCCAT AACACCATCCCCATAAACCCCATATACGTACTGCAGTCATGCTAG

Assays such as qPCR, cell enzyme linked assay (ELA), confocal microscopyand cell viability assays are performed to verify binding ofoligonucleotides to cells and identify potential cell killing propertiesof certain oligonucleotides.

Example 18 Enrichment Protocol for Microvesicles

Samples:

Experiment 1: (F98 cells: Organism: Rattus norvegicus; Cell Type:Glioblastoma; Tissue: brain; Disease: undifferentiated malignant glioma)

25 ug of microvesicles from transfected F98 cells (EGFR+) for eachpositive step

25 ug of microvesicles from parental F98 cells for each negative step

Experiment 2: (Prostate Cancer cell lines)

25 ug of microvesicles from VCaP cells for each positive step

25 ug of microvesicles from LnCaP cells for each negative step

Controls:

Reaction buffer with no microvesicles for each positive and negativestep

Reaction buffer: 1×PBS+3 mM MgCl2+0.5% F127+1 mg/ml HSA

Sample preparation: prepare 25 ug of microvesicles in 35 ul 1×PBS andadd 35 ul of 1×PBS+6 mM MgCl2+1% F127+2 mg/ml HSA to obtain 25 ug ofmicrovesicles in 70 ul of 1×PBS+3 mM MgCl2+0.5% F127+1 mg/ml HSA

Enrichment Scheme:

First round: only positive step

Second round: positive step→negative step→positive step

Third round: positive step→negative step→positive step

Fourth round: positive step→negative step→positive step

Each round ends with PCR followed by purification of ssDNA after gelelectrophoresis

I. Preparations

a) Pre-chill tabletop centrifuge at 4° C.

b) Add competitors as listed below to each 25 ug of transfected F98 orVCaP microvesicles in 70 ul reaction buffer and incubate for 20 min withshaking at 500 rpm on mixmate.

c) Mixture of two competitors:

Competitor salmon DNA: add 800 ng of salmon DNA (Stock 10 ug/ul→Dilute1:125 with reaction buffer [80 ng/ul]→10 ul input).

Competitor t-RNA: add 800 ng of tRNA (Stock 10 ug/ul→Dilute 1:125 withreaction buffer [80 ng/ul]→10 ul input).

d) Bring the volume to 180 ul with 90 ul of reaction buffer, for theblocking.

II. Library/Sample Incubation (Positive Selection)

Prepare 5 ng and 50 ng of F-TRin-35n-B Starting Library for first roundin 20 ul of reaction buffer. After heating the DNA for 3 min@95 C, putit on ice immediately for 5 min. Add DNA to the blocked cells andincubate them for 30 min at RT, with end-over-end rotation.

III. Precipitation Protocol with 6% PEG8000

a) Use microvesicles from step II as sample.

b) Add 200 ul of 12% PEG8000 to 200 ul of sample.

c) Leave sample on ice for 30 min.

d) Spin at 16000×g for 10 min and discard supernatant.

e) Re-suspend pellet in 200 ul reaction buffer and centrifuge again at16000×g for 10 min

f) Re-suspend pellet in 30 ul of reaction buffer.

g) First round: go straight to PCR. Following rounds: go to IV.

IV. Oligonucleotide Probe Elution

Add 10 ul of 0.25N NaOH to cells from III, incubate for 10 min at 50 C,mix-mate and agitate for 5-10 sec at 550 rpm→Add 10 ul of 0.25 NHCL→Spin at 16000×g for 10 min at 4 C. Collect the supernatant; thiswill be used as library for the next step.

V. Library/Sample Incubation (Negative Selection)

To each 25 ug of parental F98 or LnCaP exosomes in 70 ul reaction bufferadd the mixture of competitors (10 ul of Salmon sperm DNA@80 ng/ul+10 ulof Yeast t-RNA@80 ng/ul), bring the volume to 150 ul by addition of 60ul reaction buffer and incubate for 20 min with shaking at 500 rpm onmixmate. Then add eluted oligonucleotide probe libraries (˜50 ul) (heattreat for 3 min@95 C, incubate on ice immediately for 5 min) from stepIV and incubate 30 min at room temperature (RT).

VI. Precipitation protocol with 6% PEG8000 (using sample from III)

a) Use microvesicles/DNA mixture from step V.

b) Add 200 ul of 12% PEG8000 to 200 ul of sample.

c) Leave sample on ice for 30 min.

d) Spin at 16000×g for 10 min and collect supernatant.

VII. Library/Sample Incubation (Positive Selection)

To each 25 ug of transfected F98 or VCaP exosomes in 70 ul reactionbuffer add the mixture of competitors (10 ul of Salmon sperm DNA@80ng/ul+10 ul of Yeast t-RNA@80 ng/ul) and incubate for 20 min withshaking at 500 rpm on mixmate. Then, add oligonucleotide probe libraries(˜400 ul) (heat treat for 3 min@95 C, put it on ice immediately for 5min) from step VI and incubate 30 min at RT (total volume of 490 ul).

VIII. Precipitation Protocol with 6% PEG8000

a) Use microvesicles from step VII as sample.

b) Add 490 ul of 12% PEG8000 to 490 ul of sample.

c) Leave sample on ice for 30 min.

d) Spin at 16000×g for 10 min and discard supernatant.

e) Re-suspend pellet in 200 ul reaction buffer and centrifuge again at16000×g for 10 min

f) Re-suspend pellet in 30 ul of reaction buffer.

g) Go to PCR.

Probing

Exosomes can be probed with the enriched oligonucleotide probelibraries. For these experiments, exosomes are contacted with ssDNAenriched oligonucleotide probe libraries, precipitated with 6% PEG8000,and the co-precipitated oligonucleotide probes are amplified by qPCR.

Example 19 Oligonucleotide Enrichment on HER2+ Tissue Samples

Receptor tyrosine-protein kinase erbB-2 (ERBB2) is a protein encoded bythe ERBB2 gene, which is also frequently called HER2 (human epidermalgrowth factor receptor 2) or HER2/neu. About 20% of breast cancersoverexpress the HER2 gene, which causes cells to receive impropersignals to grow and divide. HER2+ cancers tend to be aggressive andfast-growing. For individuals with HER2+ breast cancers, the anti-HER2monoclonal antibody trastuzumab (trade name Herceptin) has been shown todramatically reduce the risk of recurrence.

In this Example, we enriched a naïve F-Trin-B oligonucleotide probelibrary against HER2 positive (HER2+) fixed tissue samples. The probelibrary is as described herein, i.e., each member has a 5′ primerCTAGCATGACTGCAGTACGT (SEQ ID NO 4) and a 3′ primerCTGTCTCTTATACACATCTGACGCTGCCGACGA (SEQ ID NO 5) surrounding a variableregion. Enrichment was performed as described herein using six rounds ofselection against FFPE (fresh frozen paraffin embedded) tissue from fivepatients with HER2+ invasive breast cancer (the HER2+ cohort) and sixpatients with HER2− invasive breast cancer (the HER2− cohort). HER2status was determined by IHC assay. The blocking buffer used duringselection comprised Salmon sperm DNA, tRNA, F127 polymer, and BSAprotein. The enrichment scheme is detailed in FIG. 15A. As indicated inthe figure, rounds 1-3 were performed using positive selection only(i.e., to enrich binders to the HER2+ samples). Rounds 4-6 used bothpositive selection against the HER2+ samples and negative selectionagainst the HER2− samples. The enriched library at the end of round 6was used in further studies as the Enriched Probing Library. Attributesof the Enriched Probing Library are shown in FIG. 15B, wherein thenumber of unique valid sequences is plotted against the numbers ofcopies of each sequence, as determined by NGS. The Enriched ProbingLibrary comprised 3.6×10⁷ unique sequences. The variable regions of the50 most prevalent sequences are listed in order of highest prevalence tolower prevalence in SEQ ID NOs 2989-3038.

The Enriched Probing Library was used to stain HER2+ and HER2− breastcancer tissue. FIG. 15C shows representative staining of a HER2+ tumorsample after no enrichment (Round 0, “R0” in the figure, left panel) orafter the six rounds of enrichment (post-Round 6, “R6” in the figure,right panel). As seen in the figure, much higher amounts of stain wereobserved with the enriched R6 probe library as compared to the naïve R0library. Similarly, FIG. 15D shows representative staining of a HER2−tumor sample after no enrichment (R0, left panel) or after the sixrounds of enrichment (R6, right panel). As seen in the figure, little tono staining was observed with either the enriched R6 probe library orthe naïve R0 library.

To identify specific members of the Enriched Probing Library that bindHER2, the experimental plan outlined in FIG. 15E was performed.Recombinant HER2 caning a histadine tag (“recHer2 His Tag”) wasconjugated to magnetic beads (“Ni magnetic beads”). The beads were mixedwith the Enriched Probing Library (“Oligonucleotide library”) andallowed to incubate to allow binding of probes to the HER2 beads. Thebound beads were washed and probe binders were recovered and identifiedby next generation sequencing (NGS) Similar experiments were performedwith negative controls to filter out probes that bind assay components,including No protein beads and Non-enriched library. A known HER2aptamer was used as a positive control. The experiments were performedtwice and a number of filters were applied, such as sequences thatappeared in both sets of binding experiments and did not bind theprotein beads. With all controls filtered out, there were 404 sequencesremaining. The variable regions of the most prevalent sequences, orderedby prevalence, are listed as SEQ ID NOs 3039-3061. These sequences areordered individually and used in an immunoassay format to identify HER2binders.

We next explored targets of the R6 library. The R6 library and theunenriched R0 library were amplified with a Biotin-C3-C6-amine primer.The libraries were TBE gel purified, conjugated with the amine reactivediazirine crosslinker, Sulfo-NHS-SS-Diazirine (Sulfo-SDAD)(sulfosuccinimidyl2-[(4,4′-azipentanamido)ethyl]-1,3′-dithioproprionate) (ThermoScientific) and purified by HPLC to remove the unconjugatedoligonucleotides. The SDAD conjugated library was then qualified forstaining HER2+ or HER2− FFPE tissue slides compared to the biotinylatedlibrary used for enrichment as detailed above.

Binding of the conjugated library was done under similar conditions usedfor the selection on HER2+ or HER2− tissue on a Ventana Discovery Ultrainstrument (Ventana Medical Systems, Tucson, Ariz.). One set of slidesfor each tissue (HER2+ or HER2) were stained with the R6 library, the R0library, or no library to assess the level of specific binding. Inparallel, nine slides per condition was removed prior to detection withStreptavidin-horse radish peroxidase (Strept-HRP) and were subjected tophoto-crosslinking 2 cm above the slide at 365 nm with a handheld UVlight for 10 minutes on ice. Slides were then scraped into QProteomebuffer without beta-mercaptoethanol added (Qiagen) and extractedaccording to manufacturer's instructions. Detergent was removed withHiPPR detergent removal columns (Thermo Fisher Scientific) and proteinconcentration was then determined with the BCA assay. Cross-linkedprotein-aptamer complexes were then affinity purified from 200 μg ofFFPE tissue lysate by incubation with 10 μL of Dynabeads® MyOne™Streptavidin C1 (Thermo Scientific) for 30 mins at room temperature,washed twice with 1×TBS, washed twice with high stringency wash buffer(10 mM Tris, 2M NaCl, 1 mM EDTA, 1% Triton X-100), followed by twowashes with low stringency wash buffer to remove the NaCl, resuspendedin 1×PBS and eluted by boiling in 1× lauryl dodecyl sulfate (LDS) withreducing agent. Reducing the samples transfers the crosslinker from theaptamer to the protein targets. The reduced samples were run underreducing conditions in a 4-12% SDS-PAGE gel at 150 volts for 15 min. Theentire lane was excised and subjected to in-gel trypsin digestion.Alkylation with iodoacetamide was replaced with Iodoacetyl Tandem MassTag™ (iodoTMT; Thermo Fisher Scientific) to facilitate identification ofcrosslinked targets by LC-MS/MS.

Results for one HER2+ case are shown in Table 28. These results werefiltered to remove proteins that also cross-linked with the unenrichedR0 library. Background binding with the R0 library appeared to be mostprevalent in the nucleus. The table indicates whether the identifiedproteins have been reported in association with HER2 or breast cancer(BrCa) or a clinical trial (for any indication), or have been used orsuggested as a drug target for any indication. References are noted inbrackets.

TABLE 28 Targets of R6 Identified for One HER2+ Tissue Sample DrugClinical Accession Gene ID Description Her2 or BrCa related? TargetTrial P60709 ACTB Actin, cytoplasmic 1 Her2 and ATPase2 in actin richmembrane domains [1] P14618 PKM Pyruvate kinase PKM Early marker forresponse to Yes Yes trastuzumab therapy-tumor M2-PK (alias for PKM)determination in the plasma of patients with metastasized breast cancercould be a helpful tool for monitoring therapeutic success.Dichloroacetate (DCA, an inhibitor of the mitochondrial pyruvatedehydrogenase kinase) was able to depolarize cancer (but not normal)mitochondria and induce apoptosis in cancer but not normal tissues [2]P17858-1 PFKL ATP-dependent 6- pentose phosphate pathway Yes Yesphosphofructokinase, liver gene; hexokinase-2, a key type mediator ofaerobic glycolysis, and the downstream proteins PFKL and ENO1; activatedby p53 [3] P25705-1 ATP5A1 ATP synthase subunit Ectopic ATP synthase isa Yes Yes alpha, mitochondrial drug target for breast cancer; ATPsynthase inhibitor citreoviridin [4], [5] P06576 ATP5B ATP synthasesubunit beta, Ectopic ATP synthase is a Yes Yes mitochondrial drugtarget for breast cancer; ATP synthase inhibitor citreoviridin [4], [5]P47914 RPL29 60S ribosomal protein L29 up in MCF-7 with treated Yes withrecombinant bromelain vs untreated MCF-7 [6] P62917 RPL8 60S ribosomalprotein L8 The RPL8 antigen may Yes represent a relevant vaccine targetfor patients with melanoma, glioma, and breast carcinoma whose tumorsexpress this protein [7] Q8TEJ3 SH3RF3 SH3 domain-containing E3 ligaseactivity; anti- Yes RING finger protein 3 apoptotic regulator for the c-Jun N-terminal kinase (JNK) pathway [8], [9] P21802 FGFR2 Fibroblastgrowth factor Target for triple negative Bca Yes Yes receptor 2therapies. Gain of function mutations in FGFRs were also identified in avariety of human cancers such as myeloproliferative syndromes,lymphomas, prostate and breast cancers as well as other malignantdiseases [10], [11] P02675 FGB Fibrinogen beta chain High plasmafibrinogen is Yes Yes correlated with poor response to trastuzumabtreatment in HER2 positive breast cancer [12] O60506 SYNCRIPHeterogeneous nuclear Component of the hepatocyte ? ribonucleoprotein Qexosomal machinery controlling microma sorting

References in Table 28

-   [1] Jeong et al., PMCA2 regulates HER2 protein kinase localization    and signaling and promotes HER2-mediated breast cancer, PNAS,    E282-E290 (pub'd online Jan. 4, 2016).-   [2] Hoopmann et al., Tumor M2 pyruvate kinase—determination in    breast cancer patients receiving trastuzumab therapy. Cancer Lett.    2002 Dec. 10; 187(1-2):223-8.-   [3] Liu et al, Comprehensive Proteomics Analysis Reveals Metabolic    Reprogramming of Tumor-Associated Macrophages Stimulated by the    Tumor Microenvironment. J Proteome Res. 2017 Jan. 6; 16(1):288-297.-   [4] Chang et al., Combination therapy targeting ectopic ATP synthase    and 26S proteasome induces ER stress in breast cancer cells, Cell    Death and Disease (2014) 5, e1540-   [5] Pan et al., ATP synthase ecto-a-subunit: a novel therapeutic    target for breast cancer, Journal of Translational Medicine 2011,    9:211-   [6] Fouz et al., Gene expression analysis in MCF-7 breast cancer    cells treated with recombinant bromelain. Appl Biochem Biotechnol.    2014 August; 173(7):1618-39.-   [7] Swoboda, et al., Shared MHC Class II—Dependent Melanoma    Ribosomal Protein L8 Identified by Phage Display. Cancer Res 2007;    67: (8). Apr. 15, 2007-   [8] Karkkainen et al., POSH2 is a RING finger E3 ligase with Rac1    binding activity through a partial CRIB domain. FEBS Lett. 2010 Sep.    24; 584(18):3867-72-   [9] Wilhelm et al., Sh3rf2/POSHER protein promotes cell survival by    ring-mediated proteasomal degradation of the c-Jun N-terminal kinase    scaffold POSH (Plenty of SH3s) protein. J Biol Chem. 2012 Jan. 13;    287(3):2247-56.-   [10] Eswarakumar et al., Cellular signaling by fibroblast growth    factor receptors. Cytokine Growth Factor Rev. 2005 April;    16(2):139-49-   [11] Wang and Guda. Integrative exploration of genomic profiles for    triple negative breast cancer identifies potential drug targets.    Medicine (Baltimore). 2016 July; 95(30):e4321.-   [12] Liu et al., High Plasma Fibrinogen is Correlated With Poor    Response to Trastuzumab Treatment in HER2 Positive Breast Cancer,    Medicine. 94(5) February 2015.-   [13] Santangelo et al., The RNA-Binding Protein SYNCRIP Is a    Component of the Hepatocyte Exosomal Machinery Controlling MicroRNA    Sorting. Cell Rep. 2016 Oct. 11; 17(3):799-808.

This Example presents an approach wherein oligonucleotide probes can beidentified using tissue sample input. The resulting oligonucleotides canbe used to identify HER2+ tissue samples and distinguish HER2− tissues.General applications of this approach include without limitationidentifying predictive biomarkers and identifying drug targets. In thisspecific case, oligonucleotide probes are identified that indicateaggressive phenotype (i.e., HER2+ tumors are known to be aggressive) butthat can be targeted by anti-HER2 treatments. We also identified certaintargets of the R6 library in a HER2+ breast cancer sample andspecifically identified several drug targets. Moreover, theoligonucleotides themselves can be used to target these proteins, andthus comprise drug candidates at the same time they identify drugtargets.

This approach in this Example can be used for any other appropriatebiomarkers.

Example 20 Oligonucleotide Enrichment on Tissue Samples to Identify DrugResponders

This Example follows the work presented in Example 19 above. The taskwas to develop an oligonucleotide library capable of distinguishingBreast Cancer (BCa) patients that respond or not to trastuzumabtreatment. Often administered together with various chemotherapeuticagents, trastuzumab alone shows efficacy in some patients (<20%) thatoverexpress HER2 as determined by IHC assay. For purposes of thisExample, responders and non-responders are based upon each patient'stime-to-next-treatment (“TNT” or “TTNT”) as a proxy for response. FIG.16A illustrates treatment schemes for BCa along with a timelinedifferentiating between responders and non-responders. As shown thenon-responders are treated with another chemotherapy treatment withinsix months of initiation of treatment with trastuzamab. The resultingoligonucleotides can be used to predict response and direct therapy toproper population of patients.

Methodology was similar to that described above in Example 19. A generalscheme for enrichment of the naïve F-trin-35 library is shown in FIG.16B (AL=aptamer library). In the figure, responders are shown aspositive samples for selection, but enrichments were similarly performedwith non-responders as the positive samples for enrichment. Six roundsof enrichment were performed. 17 enriched oligonucleotide libraries wereobtained, wherein eight libraries were trained towards non-responder andnine libraries were trained toward responders. The 17 libraries arereferred to as libraries A-S.

The libraries were trained on a set of 20 non-responders and 20responders. Given this patient cohort, responders were defined aspatients treated for 212 days and up with trastuzamab and non-responderswere defined as those treated for 8-119 days with trastuzumab. FIG. 16Cshows staining of non-responder and responder samples usingoligonucleotide probe library A sorted by staining intensity. As can beobserved in the figure, staining was more prevalent in the non-respondersamples Summary results for staining the training samples theNon-responder selected library A and responder selected library M areshown in FIG. 16D. The variable regions of the 100000 most prevalentsequences in library A are included herein as SEQ ID NOs. 3062-103061,ordered by prevalence. The variable regions of the 100000 most prevalentsequences in library M are included herein as SEQ ID NOs. 103062-203061,also ordered by prevalence. ROC curves generated for each libraryseparately and the combined A & M libraries are shown in FIG. 16E. Jointperformance was determined using logistic regression. Severalnon-responder samples without sufficient slides were removed from thesedata. As shown in FIG. 16E, the trained library A and library M hadexcellent performance for distinguishing the responder and non-respondersamples. As a control, library A was used to stain slides from breastcancer cases that were not treated with trastuzumab. No differentialstaining patterns were observed (data not shown). FIG. 16F shows resultsof using the trained libraries to classify two small test sets withsignificant AUC values as shown. The combined AUC was 0.78, whichindicates clinical utility.

Thus, the oligonucleotide probe libraries can be used to identifytrastuzumab responder tissues instead of only HER2+ tissues percurrently available companion diagnostics.

As in Example 19 above, targets are identified that are responsible forthe response (or its absence) to trastuzumab and which can be used todevelop more efficient treatment.

Example 21 Polyligand Profiling Differentiates Cancer Patients Accordingto their Benefit of Treatment

Patient response to specific cancer treatment is dependent upon subtledifferences in tumor systems states that can be difficult to access.Highly multiplexed measurement techniques can be advantageous to assessthese perturbations. In this Example, we used polyligand profiling witholigonucleotide probe libraries as described herein to perform suchanalysis. We generated two single-stranded oligodeoxynucleotide probelibraries that distinguish between tumor tissue from breast cancerpatients who either did or did not derive benefit from trastuzumab-basedregimens. Testing of an independent sample set verified the ability ofthe libraries to differentiate patients, as assessed by calculating AUCvalues from ROC curves in comparison to standardHER2-immunohistochemical scoring. Kaplan-Meier plots confirmed thatoutcomes were in accordance with test results: Test-positive patientshad a median duration of 604 days of trastuzumab treatment, whereastest-negative patients had a median duration of 129 days of trastuzumabtreatment. Thus, this Example demonstrates use of polyligand profilingto classify distinct clinical outcomes.

Precision oncology seeks to adapt the treatment of individual cancerpatients to the specific mutations and unique molecular interactionnetworks of each tumor. The genomic complexity and heterogeneity of theunderlying molecular signatures is vast and each tumor can have itsunique profile, even within the same patient (1-3). Patients thatbenefit from specific treatment regimens often differ from those that donot in features other than merely the expression status of single orsmall numbers of biomarkers. Consequently, the current paradigms forprecision oncology rely on three major pillars: First, theidentification of mutations by DNA/RNA sequencing in patients' tumors;second, the quantification of known biomarkers in tumor tissue; third,the identification of new biomarkers and master regulator genes.However, based on the limited success of several recent clinical trials,the prospect and potential of precision oncology has recently beenchallenged (4, 5). Thus, limitations have become evident and call for aparadigm-shift to facilitate the identification of patients who will orwill not benefit from a particular cancer treatment (6-8) and thedevelopment of improved companion diagnostic tests (CDx) (9). Becausesingle or small numbers of biomarkers may not always adequately predicttreatment benefit, in these cases a more effective CDx wouldsimultaneously access the multitude of intricate molecular features thatunderlie the vastly complex and currently largely unpredictablephenotype of drug response. Accessing the phenotypic diversity conferredby tumor heterogeneity and the changes in the tumor microenvironmentthat are induced by tumor cells may require identification of subtlevariations in the underlying molecular interaction network, orinteractome, the complexity of which is estimated to consist of severalhundred thousand, or even millions of multi-molecular complexes (10).Such identification may benefit from an unbiased, hypothesis-freeapproach to access informative and highly complex molecularcharacteristics, a challenge that could require an equal or greaternumber of potential detector molecules.

Here we report one approach to achieve this comprehensive goal:polyligand profiling. In this Example, we assessed the ability ofpolyligand profiling to differentiate between patients who do or do notderive benefit from a particular cancer treatment, which may also bereferred to as responders or non-responders, respectively. We thencompare polyligand profiling to immunohistochemical (IHC) analysis ofHER2, one of the most successful and widely applied CDx. Her2 isoverexpressed in fifteen to thirty percent of human breast cancers (11,12). Because HER2 amplification is associated with increased tumor cellproliferation and poor prognosis, patients with HER2 positive (HER2+)breast cancer are candidates for treatment with trastuzumab. However,only 30% of patients with HER2+ breast cancer benefit from trastuzumabmono-therapy (13). Trastuzumab (Herceptin) is a monoclonal antibody thatbinds human epidermal growth factor receptor 2 (HER2, ErbB2) andinhibits its function (14-16).

Materials and Methods for the experiments described in this Example areprovided in Example 22 below. Clinical information for patients includedin this Example is provided in Tables 29-32. In the tables, “CPP” standsfor cyclophosphamide. All samples were breast carcinomas.

TABLE 29 Patient Information: Training toward Non-Benefiters (NB)Enrichment HER2 HER2 First line Next line Additional case ID TTNT ISHIHC regimen regimen treatments Clinical TL-NB+ 158 + + carboplatinrecombinant pegylated liposomal Diagnosis: Ultrasound- paclitaxelinterferon alfa-2b doxorubicin guided core biopsy, trastuzumabpertuzumab hydrochloride right breast: Infiltrating gemcitabinecarcinoma involving all hydrochloride cores. cisplatin Stage: Unknownvinorelbine tartrate Grade: 3/Poorly fluorouracil differentiatederibulin mesylate TL-NB− 335 + + carboplatin Diagnosis: Left breast,docetaxel lumpectomy: Invasive trastuzumab ductal carcinoma. Stage: IGrade: 2/Moderately differentiated TL-NB− 280 + + carboplatinado-trastuzumab Diagnosis: Left breast paclitaxel emtansine and axilla(modified trastuzumab mastectomy): Invasive ductal carcinoma, poorlydifferentiated. Stage: IIIC Grade: 3/Poorly differentiated Alt. lib. 1+119 − − trastuzumab eribulin mesylate trastuzumab vinorelbinenab-paclitaxel tartrate Alt. lib. 1− 321 − − trastuzumab vinorelbinetartrate Alt. lib. 1− 225 + + trastuzumab ado-trastuzumab docetaxelvinorelbine emtansine pertuzumab tartrate nab-paclitaxel Alt. lib. 2+21 + + trastuzumab docetaxel carboplatin Alt. lib. 2− 294 + +trastuzumab carboplatin ado-trastuzumab gemcitabine emtansinehydrochloride Alt. lib. 2− 349 + − trastuzumab Alt. lib. 3+ 15 + +trastuzumab docetaxel carboplatin nab-paclitaxel Alt. lib. 3− 294 + +trastuzumab carboplatin ado-trastuzumab gemcitabine emtansinehydrochloride Alt. lib. 3− 349 + − trastuzumab Alt. lib. 4+ 109 + +paclitaxel cyclophosphamide pertuzumab doxorubicin trastuzumabhydrochloride Alt. lib. 4− 263 + + docetaxel pertuzumab trastuzumab Alt.lib. 4− 217 − +/− nab-paclitaxel letrozole pertuzumab fulvestranttrastuzumab Alt. lib. 5+ 56 − +/− nab-paclitaxel carboplatinado-trastuzumab pertuzumab gemcitabine emtansine trastuzumabhydrochloride eribulin mesylate Alt. lib. 5− 263 + + docetaxelpertuzumab trastuzumab Alt. lib. 5− 217 − +/− nab-paclitaxel letrozolepertuzumab fulvestrant trastuzumab Alt. lib. 6+ 56 + +/− trastuzumabcarboplatin ado-trastuzumab vinorelbine emtansine tartrate trastuzumabfulvestrant cyclophosphamide methotrexate fluorouracil leuprolideacetate docetaxel gemcitabine hydrochloride Alt. lib. 6− 321 − −trastuzumab vinorelbine tartrate Alt. lib. 6− 225 + + trastuzumabado-trastuzumab docetaxel vinorelbine emtansine pertuzumab tartratenab-paclitaxel Alt. lib. 7+ 69 n.p. − docetaxel leuprolide acetatedocetaxel pertuzumab trastuzumab Alt. lib. 7− 263 + + docetaxelpertuzumab trastuzumab Alt. lib. 7− 217 − +/− nab-paclitaxel letrozolepertuzumab fulvestrant trastuzumab

TABLE 30 Patient Information: Training toward Benefiters (B) EnrichmentHER2 HER2 First line Next line Additional case ID TTNT ISH IHC regimenregimen treatments Clinical Alt. lib. 8+ 364 + + trastuzumabado-trastuzumab vinorelbine emtansine tartrate Alt. lib. 8− 33 + +trastuzumab docetaxel vinorelbine tartrate Alt. lib. 8− 85 − −trastuzumab docetaxel docetaxel vinorelbine investigational agenttartrate cyclophosphamide fluorouracil Alt. lib. 9+ 369 − Equivocaltrastuzumab docetaxel ado-trastuzumab pertuzumab emtansine Alt. lib. 9−56 n.p. + trastuzumab leuprolide vinorelbine tartrate acetate eribulinmesylate bevacizumab nab-paclitaxel ado-trastuzumab emtansinetrastuzumab Alt. lib. 9− 20 − − trastuzumab nab-paclitaxel vinorelbinetartrate gemcitabine hydrochloride TL-B+ 371 + + carboplatin Diagnosis:Left breast, docetaxel lumpectomy: trastuzumab Infiltrating ductalcarcinoma, poorly differentiated/grade 3. Stage: IIIA Grade: 3/Poorlydifferentiated TL-B− 42 + + carboplatin pegylated cyclophosphamideDiagnosis: Right nab-paclitaxel liposomal breast mass, excision:trastuzumab doxorubicin Multifocal invasive hydrochloride ductalcarcinoma. Stage: IV Grade: 3/Poorly differentiated TL-B− 91 + +carboplatin letrozole Diagnosis: Breast, left, docetaxel Mammotomebiopsy: trastuzumab Infiltrating ductal carcinoma, no special type. Highcombined histologic grade (3, 3, 2). Stage: I Grade: 3/Poorlydifferentiated Alt. lib. 10+ 406 + + trastuzumab Alt. lib. 10− 56 n.p. +trastuzumab leuprolide vinorelbine tartrate acetate eribulin mesylatebevacizumab nab-paclitaxel ado-trastuzumab emtansine trastuzumab Alt.lib. 10− 20 − − trastuzumab nab-paclitaxel vinorelbine tartrategemcitabine hydrochloride Alt. lib. 11+ 598 + + trastuzumab vinorelbinetrastuzumab vinorelbine tartrate gemcitabine tartrate trastuzumabhydrochloride docetaxel pertuzumab eribulin mesylate Alt. lib. 11−33 + + trastuzumab docetaxel vinorelbine tartrate Alt. lib. 11− 85 − −trastuzumab docetaxel docetaxel vinorelbine investigational agenttartrate cyclophosphamide fluorouracil Alt. lib. 12+ 604 − − carboplatingemcitabine docetaxel hydrochloride trastuzumab Alt. lib. 12− 42 + +carboplatin pegylated cyclophosphamide nab-paclitaxel liposomaltrastuzumab doxorubicin hydrochloride Alt. lib. 12− 91 + + carboplatinletrozole docetaxel trastuzumab Alt. lib. 13+ 623 + + nab-paclitaxelgemcitabine ado-trastuzumab trastuzumab hydrochloride emtansine Alt.lib. 13− 67 + + docetaxel pertuzumab fulvestrant trastuzumab Alt. lib.13− 70 +/− − paclitaxel nab-paclitaxel cyclophosphamide trastuzumabdoxorubicin hydrochloride docetaxel pertuzumab ado-trastuzumab emtansinedocetaxel trastuzumab pertuzumab Alt. lib. 14+ 391 + + trastuzumab Alt.lib. 14− 56 n.p. + trastuzumab leuprolide vinorelbine tartrate acetateeribulin mesylate bevacizumab nab-paclitaxel ado-trastuzumab emtansinetrastuzumab Alt. lib. 14− 20 − − trastuzumab nab-paclitaxel vinorelbinetartrate gemcitabine hydrochloride Alt. lib. 15+ 1202 − − paclitaxelnab-paclitaxel trastuzumab gemcitabine hydrochloride Alt. lib. 15−67 + + docetaxel pertuzumab fulvestrant trastuzumab Alt. lib. 15− 70 +/−− paclitaxel nab-paclitaxel cyclophosphamide trastuzumab doxorubicinhydrochloride docetaxel pertuzumab ado-trastuzumab emtansine docetaxeltrastuzumab pertuzumab

TABLE 31 Patient Information: Test Herceptin TTNT ID Type Diagnosis TTNTCensored First line Next line Stage Grade Specimen cite Age HER2 IHCHER2 ISH ER ALx-R-18 R Mid chest wall, biopsy: 219 Y trastuzumab IVGrade 3/Poorly Chest, NOS 58 NA + Poorly differentiated nab-differentiated carcinoma, involving paclitaxel dermis and subcutaneousfibroadipose tissue. ALx-NR-10 NR Liver; biopsy: 78 N gemcitabinecarboplatin IV Unknown Liver 52 − − − Malignant epithelial hydrochlorideneoplasm, consistent trastuzumab withn metastatic docetaxel invasivelobular carcinoma. ALx-R-20 R Left breast mass, 391 Y trastuzumabUnknown Grade 3/Poorly Breast 60 + + − excision: Invasive differentiatedductal carcinoma, grade 3. ALx-R-10 R Breast, left, 3 o'clock, 7 287 Ygemcitabine Unknown Grade 3/Poorly Breast 52 − +/− + cm from nippledhydrochloride differentiated (ultrasound guided core biopsy):carboplatin Invasive ductal carcinoma. trastuzumab ALx-R-19 R Mass, leftaxilla, needle 321 Y trastuzumab IV Grade 3/Poorly Connective & Soft 46− − + biopsy: vinorelbine differentiated Tissue Invasive adenocarcinoma,tartrate morphologically consistent with recurrent breast carcinoma.ALx-R-14 R Left breast, mastectomy: 765 N fulvestrant IV Grade 3/PoorlyBreast 65 − +/− + In situ intraductal and carboplatin differentiatedinfiltrating duct carcinoma. docetaxel Tumor emboli in dermaltrastuzumab lymphatics of skin and nipple. ALx-NR-20 NR Lymph node,right axillary 70 N trastuzumab nab- IV Grade 3/ Lymph Nodes 40 − +/− +(needle core biopsy): paclitaxel paclitaxel Poorly Metastatic high-gradecarcinoma, differentiated consistent with breast primary. ALx-NR-18 NRLiver mass, biopsy: 67 N trastuzumab pertuzumab IV Grade 2/ModeratelyLiver 63 + + + Metastatic adenocarcinoma, docetaxel differentiatedcompatible with breast primary. ALx-R-5 R Left breast, 2:00, lumpectomy:339 Y CPP I Grade 2/Moderately Breast 52 + + + Infiltrating ductaldocetaxel differentiated carcinoma, moderately trastuzumabdifferentiated, grade II/III. ALx-NR-7 NR Right supraclavicular lymphnode: 56 N trastuzumab gemcitabine IIIC Unknown Lymph Nodes 47 +/− − +Single lymph node (3 cm), nab- hydrochloride replaced by metastaticcarcinoma. paclitaxel carboplatin pertuzumab ALx-R-4 R Liver, partialhepatectomy, section 5: 294 N trastuzumab gemcitabine IV Unknown Liver54 + + + Metastatic invasive mammary carcinoma. hydrochloridecarboplatin ALx-R-16 R Right breast, 8:00 N + 10, biopsy: 272 Ntrastuzumab fulvestrant IV Grade 2/Moderately Breast 49 − − + Invasiveductal carcinoma, no docetaxel differentiated special type (1.1 cm).carboplatin ALx-R-8 R Breast, right, core biopsy: 238 Y trasnizumab IGrade 3/Poorly Breast 55 + + − Infiltrating ductal carcinoma.differentiated ALx-R-17 R Left breast mass, core biopsy: 623 Ntrastuzumab gemcitabine Unknown Grade 3/Poorly Breast 46 + + − Poorlydifferentiated nab- hydrochloride differentiated infiltrating ductalcarcinoma. paclitaxel ALx-R-9 R Left breast mastectomy: 604 Ncarboplatin gemcitabine IIIC Grade 2/Moderately Breast 82 − − + Invasivelobular carcinoma. docetaxel hydrochloride differentiated trasnizumabALx-R-15 R Left breast mass: 406 Y trasnizumab IV Grade 3/Poorly Breast,35 + + + Infiltrating ductal differentiated NOS carcinoma, Nottinghamcombined histologic grade III. ALx-NR-9 NR Metastatic carcinoma 82 Ntrasnizumab CPP I Grade 2/Moderately Axillary 46 + + − involving one outof pertazumab doxorubicin differentiated lymph seventeen (1/17)trasnizumab hydrochloride node axillary lymph nodes. docetaxel ALx-R-11R Right breast, “masses” 1202 N trasnizumab gemcitabine IV Grade3/Poorly Breast, 79 − − + at 10 and 2 o'clock paclitaxel hydrochloridedifferentiated NOS positions, resection: nab-paclitaxel Invasive lobularcarcinoma. ALx-R-7 R 369 N trasnizumab pertuzumab II Grade 3/PoorlyBreast, 52 +/− − − docetaxel differentiated NOS ALx-NR-14 NR Peritonealmass, needle 69 N trasnizumab leuprolide IV Unknown Peritoneal 35 − − +core biopsy: docetaxel acetate cavity Metastatic carcinoma, withpertuzumab morphologic and immunohistochemical features consistent withthe patient's known breast center. ALx-R-13 R Skin, “lesion” on right598 N trasnizumab trasnizumab IV Grade 3/Poorly Skin of 62 + + −anterior chest w all, vinorelbinc vinorelbine differentiated chest wallexcisional biopsy: tartrate tartrate Metastatic carcinoma of mammaryorigin with prominent apocrine features. ALx-R-1 R Left axillarysentinel 301 Y fluorouracil Unknown Grade 3/Poorly Breast, 66 +/− +/− −nodes, left breast: CPP differentiated NOS Invasive ductal carcinoma.methotrexate Nottingham grade 3. trasnizumab ALx-NR-12 NR Right chestwall mass excision: 109 N trasnizumab CPP I Grade 2/Moderately Chestwall, 53 + + − Invasive ductal carcinoma, paclitaxel doxorubicindifferentiated NOS grade 2. pertuzumab hydrochloride ALx-R-6 R Breast,right, mastectomy: 291 Y trasnizumab II Grade 2/Moderately Breast, 56 −− + Infiltrating mammary docetaxel differentiated NOS carcinoma withmixed CPP ductal and lobular phenotype. ALx-NR-11 NR Rightadrenalectomy: 56 N vinorelbine carboplatin IV Unknown Adrenal 54+/− + + Adenocarcinoma, consistent with tartrate gland, metastaticmammary carcinoma. trastuzumab NOS ALx-NR-4 NR Lymph node, right 113 Nnab- capecitabine IIIA Grade 1/Well Axillary 51 +/− − − axillary,excision: paclitaxel differentiated lymph Metastatic adenocarcinomacarboplatin node consistent with breast primary. pertuzumab trastuzumabALx-R-41 R Left breast, simple mastectomy: 190 Y pertuzumab II Grade3/Poorly Breast, 34 +/− − + Invasive ductal carcinoma, docetaxeldifferentiated NOS Nottingham grade III. carboplatin trastuzumabALx-R-42 R Right breast, core biopsy: 196 Y trastuzumab I Grade 3/PoorlyBreast 52 − − + Infiltrating ductal carcinoma. pertuzumab differentiateddocetaxel ALx-R-43 R Right breast, simple mastectomy: 375 Y trastuzumabII Grade 3/Poorly Breast 41 + + − Recurrent invasive grade 3 docetaxeldifferentiated ductal carcinoma. carboplatin trastuzumab ALx-R-44 R 334Y trastuzumab IV Unknown Liver 64 − + + ALx-R-45 R Left breast scarrevision: 406 Y trastuzumab I Grade 3/Poorly Breast 54 − + + Invasivepoorly-differentiated fulvestrant differentiated ductal carcinoma.ALx-R-47 R Left breast inframammary crease: 546 Y trastuzumab I UnknownSkin 55 + + − Metastatic carcinoma involving dermal lymphovascularspaces. ALx-R-48 R Left breast and axillary 856 Y trastuzumab IV Grade3/Poorly Axillary 40 − +/− − contents, modified differentiated lymphradical mastectomy: node Invasive ductal carcinoma. ALx-R-49 NR Liver,biopsy: 106 N trastuzumab bevacizumab IV Grade 3/Poorly Liver 46 + + +Metastatic carcinoma. carboplatin differentiated ALx-R-50 R Skin, leftbreast, biopsy: 186 N trastuzumab vinorelbine I Unknown Connective &Soft 41 + + − Dermal intralymphatic tumor, tartrate Tissue consistentwith inflammatory carcinoma. ALx-R-51 R Right axillary/pectoral 196 Ntrastuzumab vinorelbine I Grade 3/Poorly Connective & Soft 55 + + +tumor, excision: tartrate differentiated Tissue Infiltrating ductalcarcinoma trastuzumab involving subcutaneous fibroadipose tissue andadjoining skeletal muscle. ALx-R-52 NR Liver, segment VI, 80 Ntrastuzumab octreotide IV Unknown Liver 46 + + − partial resection:pertuzumab acetate Consistent with fulvestrant metastatic mammarycarcinoma. ALx-R-53 R Right liver lobe, partial lobectomy: 190 Ntrastuzumab fulvestrant IV Unknown Liver 63 − − + Metastatic carcinoma,consistent with breast primary. ALx-R-54 R Left breast mass: 433 Nleuprolide paclitaxel IV Grade 3/Poorly Breast 44 QNS Infiltrating ductacetate differentiated carcinoma with lobular docetaxel features, grade3. trastuzumab pertuzumab ALx-R-55 R Pelvic mass, biopsy: 183 Ntrastuzumab nab-paclitaxel IV Unknown Pelvis, 42 +/− + + Metastaticcarcinoma. vinorelbine NOS tartrate ALx-R-56 R Liver, needle biopsy 250N trastuzumab ado- IV Unknown Liver 68 + + − (SS-13-01227): pertuzumabtrastuzumab Mild steatosis and cholestasis. docetaxel emtansine ALx-R-57NR Breast, left, simple mastectomy: 49 N trastuzumab ado- II Grade2/Moderately Breast. 44 + + − Residual infiltrative mammary trastuzumabdifferentiated NOS carcinoma with ductal emtansine and lobular features.ALx-R-58 NR SU-12-02676: Right 154 N trastuzumab docetaxel Unknown Grade3/Poorly Breast, 41 − − − breast (lumpectomy): differentiated NOSResidual poorly differentiated adenocarcinoma, multifocal. ALx-R-59 NRRight axillary mass: 128 N trastuzumab docetaxel Unknown Grade 3/PoorlyBreast, 62 + + − High grade infiltrating differentiated NOS ductalcarcinoma. ALx-R-60 NR Bone, right distal femur 129 N trastuzumabpertuzumab IV Unknown Femur 51 + + + tumor, excision: docetaxelMetastatic adenocarcinoma, compatible with breast primary.

TABLE 32 Patient Information: Test Platinum/Taxane TTNT ID TypeDiagnosis TTNT Censored First line Next line Stage Grade Specimen citeAge HER2 IHC HER2 ISH ER Pl-1 NR Right breast and medial 22 N docetaxelCPP Unknown NA Breast 73 − − − portion of inframammary bridge, simplemastectomy: Infiltrating ductal carcinoma, grade 2 (Nottinghamclassification). Pl-2 R Left masectomy breast tissue: 822 Y nab- IIIAGrade 2/Moderately Breast 49 − − + Invasive mammary carcinoma.paclitaxel differentiated fluorouracil Pl-3 R Right breast (simplemastectomy): 461 N nab- gemcitabine II Grade 3/Poorly Breast 43 − − +Infiltrating ductal carcinoma. paclitaxel hydrochloride differentiatedbevacizumab carboplatin Pl-4 NR Lymph node from left neck (biopsy): 21 Nnab- eribulin Unknown NA Lymph Nodes 40 − +/− + Metastatic poorlydifferentiated paclitaxel mesylate carcinoma involving one out of onelymph node’ (1/1). Pl-5 NR Right axillary lymph node (biopsy): 40 Ndocetaxel doxorubicin I Unknown Lymph Nodes 43 − − − Metastaticcarcinoma. hydrochloride CPP Pl-6 NR Liver “mass” (percutaneous 29 Ncarboplatin gemcitabine IV Grade 2/Moderately Liver 47 − others − needlecore biopsies): nab- hydrochloride differentiated Metastaticadenocarcinoma, paclitaxel consistent with breast primary. bevacizumabPl-7 NR Touch preps and core 84 N nab- patupilone IV Unknown Bones &Joints 60 − − + biopsy, left iliac crest: paclitaxel Positive formalignancy. Pl-8 NR Breast, left, 12-3:00: 62 N docetaxel doxorubicin IIGrade 3/Poorly Breast 62 − − + Invasive lobular carcinoma, CPPhydrochloride differentiated pleomorphic type. Pl-9 NR Mass,retroperitoneum; biopsy: 59 N nab- patupilone IV Unknown Retroperitoneum55 − − + Metastatic carcinoma, consistent paclitaxel & Peritoneum withbreast primary. Pl-10 NR Left breast (182 grams), simple 98 N paclitaxelCPP II Grade 3/Poorly Breast 39 − − − mastectomy: doxorubicindifferentiated Infiltrating ductal carcinoma. hydrochloride fluorouracilPl-11 NR Left breast, simple mastectomy: 70 N docetaxel fluorouracil IVGrade 2/Moderately Breast 44 − − + Infiltrating lobular carcinoma.patupilone differentiated Pl-12 R Lymph nodes, right axillary, 402 Npaclitaxel gemcitabine IV Grade 2/Moderately Lymph Nodes 56 − − −excision: bevacizumab hydrochloride differentiated Metastaticadenocarcinoma consistent nab- with breast primary involving onepaclitaxel of three lymph nodes (1/3). Pl-13 R Ascites fluid, cytology:631 N gemcitabine gemcitabine IV Unknown Peritoneal Fluid 70 − − +Metastatic adenocarcinoma, consistent hydrochloride hydrochloride withlobular breast carcinoma. docetaxel Pl-14 NR Left breast, needlebiopsies: 56 N nab- doxonibicin IIIA Grade 3/Poorly Breast 62 − − −Invasive ductal carcinoma, paclitaxel hydrochloride differentiated grade3 (of 3). CPP Pl-15 R Core needle biopsy, left lateral 449 N CPPeribulin IV Grade 3/Poorly Lymph Nodes 73 − − − breast/axillary lymphnode mass: epirubicin mesylate differentiated Metastatic poorlydifferentiated hydrochloride adenocarcinoma, morphologically paclitaxeland immunohistochemically compatible with primary ductal breast origin.Pl-16 R Right breast mass, excision: 343 N doxorubicin gemcitabine IIGrade 3/Poorly Breast 44 − − − Invasive ductal carcinoma. hydrochloridehydrochloride differentiated CPP docetaxel Pl-17 NR Supraclavicular nodeleft: 99 N docetaxel gemcitabine IV Unknown Lymph Nodes 57 − − − Lymphnode totally replaced by carboplatin hydrochloride metastatic carcinoma,consistent with primary breast carcinoma. The tumor extends completelythrough the node capsule to the surrounding fatty tissues. Pl-18 R Chestwall mass, right 341 N carboplatin eribulin IV Grade 2/ModeratelyConnective & 80 +/− − + posterior, biopsy: docetaxel mesylatedifferentiated Soft Tissue Metastatic carcinoma, favor mammary primary.Pl-19 NR Reamings, hip, right, biopsy: 105 N paclitaxel gemcitabine IVGrade 3/Poorly Bones & Joints 64 − − − Metastatic poorly differentiatedcarboplatin hydrochloride differentiated carcinoma consistentbevacizumab with breast origin. Pl-20 R Right breast, 10 o'clock,lumpectomy: 674 Y CPP IIIA Grade 3/Poorly Breast 69 − − + Infiltratingductal adenocarcinoma. docetaxel differentiated Pl-21 NR Liver, needlebiopsies: 21 N docetaxel doxorubicin IV Grade 2/Moderately Liver 45 +/−− + Metastatic carcinoma morphologically hydrochloride differentiatedconsistent with breast primary. Pl-22 NR Left breast lumpectomy, 2o'clock: 49 N docetaxel gemcitabine IIIC Grade 3/Poorly Breast 71 − − −Infiltrating ductal adenocarcinoma, CPP hydrochloride differentiatedElston grade III, 4 cm maximum dimension, present 1 mm from the inferiormargin. Pl-23 R Left breast mass, additional 686 Y docetaxel II Grade3/Poorly Breast 72 − − − margins left breast, axillary therapeuticdifferentiated contents, and additional immune axillary contents:globulin Invasive lobular carcinoma. CPP Pl-24 NR Breast, right,Mammotome biopsy: 22 N nab- pegylated I Grade 3/Poorly Breast, NOS 43 −− − Infiltrating ductal carcinoma, paclitaxel liposomal differentiatedno special type carboplatin doxonibicin hydrochloride Pl-25 NR Duodenum,biopsy: 63 N carboplatin capecitabine IV Grade 3/Poorly Duodenum 31 +/−− − Poorly-differentiated carcinoma, gemcitabine differentiatedcompatible with metastatic hydrochloride mammary carcinoma. Pl-26 NRRight breast mass core needle 106 N paclitaxel IV Grade 3/Poorly Breast,NOS 56 − − − biopsies (image directed): differentiated Infiltrating ductcarcinoma, high grade (grade 3). Pl-27 NR Right breast mass: 43 Ncarboplatin methotrexate Unknown NA Breast, NOS 59 − − − Invasive ductalcarcinoma. docetaxel Nottingham grade 3. Pl-28 NR Left breast, modified70 N carboplatin Methotrexate IIIA Grade 3/Poorly Breast, NOS 61 − − −radical mastectomy fluorouracil differentiated with level I/II nodedissection: 3.5 cm poorly differentiated invasive ductal carcinoma withfocal necrosis and foci of high grade ductal carcinoma in situ (comedotype). Pl-29 NR Left breast mass, left 70 N nab- gemcitabine UnknownGrade 3/Poorly Breast, NOS 53 − − − breast core biopsy: paclitaxelhydrochloride differentiated Infiltrating ductal carcinoma. carboplatinPl-30 NR Left breast Mammotome biopsy: 83 N nab- pegylated II Grade3/Poorly Breast, NOS 58 − − − Infiltrating carcinoma. paclitaxelliposomal differentiated carboplatin doxorubicin hydrochloride Pl-31 NRRight breast segment, 22 N carboplatin CPP II Grade 3/Poorly Breast. NOS62 − − − segmental mastectomy: docetaxel differentiatedPoorly-differentiated invasive ductal carcinoma. Pl-32 NR Right necknode, core biopsy: 58 N nab- eribulin Unknown NA Connective, 45 NA NA −Metastatic poorly differentiated paclitaxel mesylate subcutaneous andadenocarcinoma. other soft tissues of head, face and neck Pl-33 NR 28 Ncarboplatin pemetrexed IIIA Grade 3/Poorly Breast. NOS 57 − − −gemcitabine disodium differentiated hydrochloride Pl-34 R Breast, left,core biopsy: 630 N carboplatin pegylated IV Grade 2/Moderately Breast.NOS 78 − − − Invasive carcinoma, grade 2 (of 3). liposomaldifferentiated doxorubicin hydrochloride Pl-35 NR Left breast andaxilla, 80 N carboplatin doxorubicin IIIC Grade 3/Poorly Breast. NOS 75− − − modified radical mastectomy: gemcitabine hydrochloridedifferentiated Invasive ductal carcinoma. hydrochloride Pl-36 NR Leftbreast biopsy: 91 N nab- pegylated Unknown Grade 3/Poorly Breast. NOS 50− − − Infiltrating ductal carcinoma. paclitaxel liposomal differentiatedHigh combined histologic grade (3, 3, 3). carboplatin doxorubicinhydrochloride Pl-37 NR T9 bone, core biopsy: 56 N bevacizumab CPP IVUnknown Bone, NOS 45 +/− − + Metastatic carcinoma, paclitaxeldoxorubicin compatible with breast primary. hydrochloride Pl-38 NRBreast, left 2:00 N10 40 N carboplatin doxorubicin I Grade 3/PoorlyBreast, NOS 46 − − − (ultrasound-guided, paclitaxel hydrochloridedifferentiated vacuum-assisted biopsy): CPP Invasive ductal carcinoma,grade 3. Pl-39 NR Right breast, mastectomy: 63 N docetaxel FluorouracilII Grade 3/Poorly Breast, NOS 83 − − − Infiltrating ductal carcinoma.carboplatin CPP differentiated Pl-40 NR Left breast mass, core biopsy:84 N paclitaxel CPP IV Grade 3/Poorly Breast, NOS 37 − − − Infiltratingductal carcinoma. doxorubicin differentiated hydrochloride Pl-41 NRBreast, left (biopsies): 73 N gemcitabine doxorubicin IV Grade2/Moderately Breast, NOS 56 +/− − − Infiltrating ductal hydrochloridehydrochloride differentiated carcinoma, grade 2. carboplatin CPP

In this Example, we subjected a library of 10¹³ unique single-strandedDNA oligodeoxynucleotides (ssODNs; which may also be referred to asaptamers or oligonucleotide probes herein) (17, 18) to several rounds ofpositive and negative selection in situ for subsets of sequences thatpreferentially bind to formalin-fixed and paraffin-embedded (FFPE) tumortissue slices from breast cancer patients who did or did not derivebenefit from regimens containing trastuzumab. We refer to this processas library training. Each patient's time to next therapy (TTNT), i.e.the time that elapsed until trastuzumab-based treatment changed definedwhether a patient belonged to the group that derived benefit fromtrastuzumab containing treatment (B) or to the group that did not (NB)(19). The definition of treatment benefit (B) and no benefit (NB) byTTNT is consistent with recently published FDA guidance (20). For thepurpose of library training, cases with TTNT greater than 180 days wereconsidered as B and cases with TTNT fewer than 180 days were consideredNB. In total, 8 NB cases (see Table 29) and 9 B-cases (see Table 30)were used for the training of 17 different libraries. Among them weretwo best-performing trained libraries, TL-B and TL-NB (see “Enrichmentcase ID” in Tables 29-30), which we employed to generate a predictiveassay based on polyligand profiling that differentiates B from NBpatients.

FIGS. 17A-B show the rationale for the selection of patients (FIG. 17A)and the workflow of the training and testing procedure of the ssODNlibrary, including the number of cases used in each step (FIG. 17B).Clinical information for each of the patients included in this process,such as tumor anatomical site, treatment, hormonal status, etc. isprovided in Tables 29-32. The starting library was subjected to positiveselection using one NB case (rounds 1-6) and negative selection usingtwo B cases (rounds 4-6) to generate TL-NB, and vice versa forgenerating TL-B. In Tables 29-30, the cases are grouped in triads. Forexample, positive selection was performed using NB case, TL-NB+, andnegative selection in the same enrichment was performed using the two Bcases just below identified as TL-NB- and TL-NB-. For addition detailsof the selection schemes and protocols, see FIG. 17C and Example 22.Testing readout is based on polyligand-histochemistry (“PHC”) stainingof FFPE slides from cases independent of those used for training. Alltissue samples used for training and testing were collected prior totreatment to ensure that library training is independent of molecularchanges in the tumor tissue that occur as a result of treatment.

FIG. 17C provides an overview of the procedure for ssODN librarytraining FIG. 17Ci outlines positive training steps towards a librarythat identifies non-benefitting (NB) cases: (i) incubation of the ssODNlibrary with the NB tissue; (ii) removal of unbound sequences, (iii)dissection of tumor tissue and recovery of the subset of sequences,specific to the NB cancer tissue. SN: supernatant. Recovered ssODNs wereamplified by PCR, converted to ssODNs and used for the next traininground. The slide images on the left show tissue appearance: Slide 1)Hematoxylin and eosin (H&E) staining of NB tissue (tumor area outlinedin green); Slide 2) Nuclear Fast Red (NFR) stained tissue afterpartitioning before dissection; Slide 3) Remaining normal tissue afterdissection of cancer tissue with bound ssODNs. FIG. 17Cii outlinestraining steps with additional counter-selection steps on benefit (B)cases: (i) incubation of the ssODN library with the 1st B tissue; (ii)incubation of the supernatant from (i) with the 2nd B case; (iii)incubation of the supernatant from (ii) with the NB case from A; (iv)and (v) correspond to the steps (ii) and (iii) in A. The slide images onthe left show tissue appearance: Slide 4) Hematoxylin and eosin (H&E)staining of 1st B tissue (tumor area outlined in green); Slide 5)Hematoxylin and eosin (H&E) staining of 2nd B tissue (tumor areaoutlined in green); Slides 2 and 3 are the same as in FIG. 17Ci. FIG.17Ciii outlines the entire ssODN library training, which is comprised ofthree training rounds as shown in FIG. 17Ci (indicated “i)”); followedby three training rounds as shown in FIG. 17Cii (indicated “ii)”). FIG.17Di shows staining of the tissue from the NB-case that was used duringthe selection process with untrained library (round 0), compared to thetrained TL-NB library (round 6; upper panel: 4×, lower panel: 20×). FIG.17Dii shows staining of tissue from an NB case not used during theselection process with untrained library (round 0), compared to thetrained library TL-NB (round 6; upper panel: 4×, lower panel: 20×). FIG.17Diii shows staining of the tissue from the responder case employed forcounter selection in the training of TL-NB, using the output ssODNs fromround 3 (left), compared to the output ssODNs from round 5 (right).

In more detail, the training library for non-benefiting patients TL-NBwas obtained by incubating the starting library directly with FFPE-fixedbreast tumor tissue from a trastuzumab NB-patient. After one hour ofincubation (step i), non-binding ssODNs were removed by washing (stepii), followed by a dissection of the tumor tissue away from normaladjacent tissue (step iii) and asymmetric PCR-amplification of ssODNsbound to the dissected tumor tissue. See FIG. 17Ci and Example 22. Thesepositive selection steps were performed a total of three times on serialtissue sections of the same case (rounds 1-3; FIG. 17Ci, iii). ThessODN-library from the third round of enrichment was then subjected totwo consecutive counter-selection steps by incubating with FFPE tissuefrom two trastuzumab B-patients. The supernatant from the secondcounter-selection step, i.e. the library that is depleted of ssODNs thatare associated with binding to B-tumor tissue, was transferred to a newtissue section from the original NB-patient's tumor for one finalpositive selection step as described above and the subset of boundssODNs was PCR-amplified (FIG. 17Cii). The negative-negative-positivesteps were performed a total of three times (SELEX rounds 4-6; FIG.17Ciii). A comparison of the staining intensities of the unselectedlibrary with that of the round 6 TL-NB library on the NB case used forthe selection showed a significant increase of staining for the enrichedlibrary (FIG. 17Di, round 6), whereas no or weak staining was seen forthe unselected library (FIG. 17Di, round 0). Similar staining wasobserved on a separate NB case not used for the library training (FIG.17Dii). Not unexpectedly, staining intensity was also high when thePCR-amplified library from round 3 (the enriched library before counterselection) was applied to a B case (FIG. 17Diii, left panel). However,after round 5 a notable decrease in staining intensity was observed forTL-NB applied to the same B case, indicating that the counter-selectionsteps were effective (FIG. 17Diii, right panel). Thus, TL-NB shows highstaining intensities on NB tissue, while it stains the B-case used forcounter selection with considerably weaker intensity. TL-NB wastherefore suitable for PHC staining on an independent test-set.

To test whether we could also train a random library for a set of ssODNsthat preferentially stain B-cases, we carried out a separate enrichmentprocess in the opposite direction, using B-cases for positive, andNB-cases for counter-selection. The resulting trained library, TL-B,showed preferential staining of B-cases compared to the NB-cases or theuntrained starting library. FIG. 17E shows staining of library TL-B on Band NB tissues. FIG. 17Ei shows staining of tissue from a B case notused during the selection process with untrained library (round 0),compared to the trained library TL-B (round 6; upper panel: 4×, lowerpanel: 20×) FIG. 17Eii shows staining of the tissue of a patient who didnot derive benefit from trastuzumab-based regimens (NB) bypolyligand-histochemistry (PHC), using the library enriched on abenefiting case (B) at round 4, compared to TL-B after round 6.

Taken together, the PHC staining characteristics observed with TL-NB andTL-B indicate sufficient selection pressure and successful enrichmenttoward their targeted phenotypes. The variable regions of the 100000most prevalent sequences in the NB selected enrichment (library TL-NB)are included herein as SEQ ID NOs. 3062-103061, ordered by prevalence.The variable regions of the 100000 most prevalent sequences in the Bselected enrichment (library TL-B) are included herein as SEQ ID NOs.103062-203061, also ordered by prevalence.

To evaluate the performance of TL-NB and TL-B on cases independent fromthose used for training, we verified that histological H-scoring of thetissue staining intensity obtained with the trained libraries can beemployed for the quantitative comparison of the cases, similar tostandard pathological practice for IHC (2l). The scoring for bothcytoplasmic and nuclear staining was performed by a board-certifiedpathologist who was blinded to the patient outcomes. Protocols are foundin FIGS. 17F-H and Example 22 and scoring results are shown in Tables33-36. Examples of PHC staining intensity levels in the cytoplasm andthe nuclei of breast cancer FFPE tissue ranging from 0 to 3 are shown inFIG. 17F. See also FIGS. 17I-J, which show polyligand histochemistry(PHC) staining profiles comparison of the non-enriched starting library(R0) and enriched libraries TL-NB and TL-B on patients not benefiting(NB) and benefiting (B) from trastuzumab containing treatment. The viewareas are matched in each row between libraries within each panel. Thelibrary R0 usually exhibits little to no staining, while the enrichedlibraries can be scored from 1+ to 3+. Library TL-NB, which was enrichedtoward a trastuzumab non-benefiting case exhibits stronger intensity onNB cases, except for NB-15. Library TL-B, which was enriched toward aTrastuzumab responder case, exhibits stronger intensity on the B cases.The magnification in FIGS. 17I-J is 20×. The histological scores werecalculated by standard methods by determining the percentages of cellson the entire tissue, classified to fall within each PHC intensity levelin the cytoplasm and the nucleus. See FIG. 17G and Example 22. Toevaluate staining and scoring reproducibility, we selected cases thatshowed weak and strong staining with TL-NB and then scored nuclearstaining (22) between technical replicates (FIG. 17H, first panel“Intra-assay”), different operators (FIG. 17H, second panel“Inter-operator”), different batches of library (FIG. 17H, third panel:Inter-batch”), and different instruments (FIG. 17H, fourth panel“Inter-instrument”). The classification of four strongly and weaklystaining cases was consistent and independent of these variables,indicating that the staining and scoring is reproducible. To furtherassess the technical reproducibility of PCR amplified versions of TL-NBand TL-B, both libraries were amplified for up to ten PCR-generations.See FIGS. 17K-L, which show technical reproducibility of the stainingwith libraries TL-NB and TL-B resulting from different PCR-generations1-5 (FIG. 17K), and 6-10 (FIG. 17L). Examples from two different cases(Her2+ and Her2−/low) at 20× magnifications are shown. For each PCRgeneration, an aliquot (0.4 ng) of each preceding library generation wasamplified for 10 PCR cycles. Each PCR generation of TL-NB and TL-B wasthen used for the staining of consecutive tissue sections from the sameNB patient for TL-NB and the same B patient for TL-B. Each PCRgeneration of TL-NB and TL-B was used for the staining of consecutivetissue sections from the same NB patient for TL-NB and the same Bpatient for TL-B. No significant difference in the staining was observedfrom generation to generation, indicating highly robust performance ofboth libraries after excessive PCR amplification.

TABLE 33 Histological Scoring, TL-NB, Trastuzamab TTNT Case ID in Her2Nuclear Cytoplasmic Nuclear Cytoplasmic Total the test set status 0 1 23 0 1 2 3 score score score ALx-R-42 Neg 80 0 20 0 0 100 0 0 40 100 140ALx-R-53 Neg 20 80 0 0 0 100 0 0 80 100 180 ALx-R-41 Neg 5 10 85 0 0 1000 0 180 100 280 ALx-R-55 Neg 30 60 10 0 0 100 0 0 80 100 180 ALx-R-58Pos 30 40 30 0 0 70 30 0 100 130 230 ALx-R-60 Pos 15 30 55 0 100 0 0 0140 0 140 ALx-R-59 Pos 20 50 30 0 30 50 20 0 110 90 200 ALx-R-50 Neg 1000 0 0 30 70 0 0 0 70 70 ALx-R-51 Pos 5 90 5 0 0 100 0 0 100 100 200ALx-R-43 Pos 1 20 79 0 0 100 0 0 178 100 278 ALx-NR-4 Neg 10 90 0 0 0100 0 0 90 100 190 ALx-NR-7 Neg 60 40 0 0 80 20 0 0 40 20 60 ALx-NR-9Pos 20 80 0 0 0 100 0 0 80 100 180 ALx-NR-10 Neg 30 40 30 0 0 60 40 0100 140 240 ALx-NR-11 Pos 50 40 10 0 30 70 0 0 60 70 130 ALx-NR-12 Pos20 50 30 0 0 100 0 0 110 100 210 ALx-NR-14 Neg 10 0 80 10 0 100 0 0 190100 290 ALx-NR-18 Pos 60 20 20 0 0 90 10 0 60 110 170 ALx-NR-20 Neg 9010 0 0 0 80 20 0 10 120 130 ALx-R-1 Neg 40 60 0 0 5 20 75 0 60 170 230ALx-R-4 Pos 100 0 0 0 0 40 60 0 0 160 160 ALx-R-5 Pos 60 40 0 0 5 85 100 40 105 145 ALx-R-6 Neg 10 60 30 0 10 90 0 0 120 90 210 ALx-R-7 Neg 4060 0 0 100 0 0 0 60 0 60 ALx-R-8 Pos 95 5 0 0 99 1 0 0 5 1 6 ALx-R-9 Neg50 50 0 0 50 50 0 0 50 50 100 ALx-R-10 Neg 60 40 0 0 0 80 20 0 40 120160 ALx-R-11 Neg 90 10 0 0 30 70 0 0 10 70 80 ALx-R-13 Pos 90 10 0 0 9010 0 0 10 10 20 ALx-R-14 Neg 90 10 0 0 10 90 0 0 10 90 100 ALx-R-15 Pos100 0 0 0 10 85 5 0 0 95 95 ALx-R-16 Pos 100 0 0 0 0 100 0 0 0 100 100ALx-R-17 Neg 100 0 0 0 10 50 40 0 0 130 130 ALx-R-18 Pos 15 85 0 0 0 8020 0 85 120 205 ALx-R-19 Neg 95 5 0 0 10 90 0 0 5 90 95 ALx-R-20 Pos 5040 10 0 20 70 10 0 60 90 150 ALx-R-45 Neg 20 60 20 0 100 0 100 ALx-R-44Neg 80 20 0 0 20 0 20 ALx-R-48 Neg 0 60 40 0 140 0 140 ALx-R-47 Neg 0100 0 0 100 0 100 ALx-R-54 Pos 100 0 0 0 0 0 0 ALx-R-56 Pos 20 70 10 090 0 90 ALx-R-52 Neg 20 80 0 0 80 0 80 ALx-R-57 Pos 0 50 50 0 150 0 150ALx-R-49 Pos 90 10 0 0 10 0 10

TABLE 34 Histological Scoring, TL-NB, Platinum/Taxane TTNT Case ID inHer2 Nuclear Cytoplasmic Nuclear Cytoplasmic Total the test set status 01 2 3 0 1 2 3 score score score Pl-1 Neg 5 45 50 0 0 100 0 0 145 100 245Pl-2 Pos 70 30 0 0 10 70 20 0 30 110 140 Pl-3 Neg 10 55 35 0 0 100 0 0125 100 225 Pl-4 Neg 30 70 0 0 0 100 0 0 70 100 170 Pl-5 Neg 45 50 5 0 0100 0 0 60 100 160 Pl-6 Pos 20 40 40 0 0 100 0 0 120 100 220 Pl-7 Neg 6030 10 0 10 90 0 0 50 90 140 Pl-8 Pos 35 45 20 0 0 70 30 0 85 130 215Pl-9 Pos 0 0 0 Pl-10 Neg 0 10 90 0 100 0 0 0 190 0 190 Pl-11 Pos 5 90 50 0 100 0 0 100 100 200 Pl-12 Neg 20 40 40 0 10 90 0 0 120 90 210 Pl-13Pos 50 40 10 0 0 100 0 0 60 100 160 Pl-14 Pos 80 20 0 0 50 50 0 0 20 5070 Pl-15 Neg 0 0 0 Pl-16 Neg 20 80 0 0 0 60 40 0 80 140 220 Pl-17 Neg 2080 0 0 100 0 0 0 80 0 80 Pl-18 Neg 0 0 0 Pl-19 Neg 0 0 0 Pl-20 Neg 10 900 0 0 100 0 0 90 100 190 Pl-21 Pos 5 10 85 0 100 0 0 0 180 0 180 Pl-22Pos 60 30 10 0 70 30 0 0 50 30 80 Pl-23 Neg 10 30 60 0 10 90 0 0 150 90240 Pl-24 Pos 5 70 25 0 0 100 0 0 120 100 220 Pl-25 Pos 0 0 0 Pl-26 Pos10 80 10 0 0 80 20 0 100 120 220 Pl-27 Neg 10 70 20 0 20 80 0 0 110 80190 Pl-28 Pos 35 40 25 0 0 60 40 0 90 140 230 Pl-29 Pos 3 67 30 0 0 1000 0 127 100 227 Pl-30 Pos 10 80 10 0 10 90 0 0 100 90 190 Pl-31 Pos 2060 20 0 0 100 0 0 100 100 200 Pl-32 Neg 20 80 0 0 0 80 20 0 80 120 200Pl-33 Neg 40 60 0 0 100 0 0 0 60 0 60 Pl-34 Pos 70 30 0 0 20 60 20 0 30100 130 Pl-35 Pos 50 50 0 0 10 90 0 0 50 90 140 Pl-36 Pos 80 10 10 0 0100 0 0 30 100 130 Pl-37 Neg 20 10 70 0 30 70 0 0 150 70 220 Pl-38 Neg30 60 10 0 10 90 0 0 80 90 170 Pl-39 Pos 10 90 0 0 0 100 0 0 90 100 190Pl-40 Neg 60 35 5 0 20 80 0 0 45 80 125 Pl-41 Neg 5 45 50 0 0 80 20 0145 120 265

TABLE 35 Histological Scoring, TL-B, Trastuzamab TTNT Case ID in Her2Nuclear Cytoplasmic Nuclear Cytoplasmic Total the test set status 0 1 23 0 1 2 3 score score score ALx-R-42 Neg 35 45 20 0 0 100 0 0 85 100 185ALx-R-53 Neg 30 70 0 0 0 100 0 0 70 100 170 ALx-R-41 Neg 10 80 10 0 1090 0 0 100 90 190 ALx-R-55 Neg 90 10 0 0 0 100 0 0 10 100 110 ALx-R-58Pos 20 80 0 0 0 100 0 0 80 100 180 ALx-R-60 Pos 10 40 50 0 10 90 0 0 14090 230 ALx-R-59 Pos 15 85 0 0 0 100 0 0 85 100 185 ALx-R-50 Neg 50 50 00 0 100 0 0 50 100 150 ALx-R-51 Pos 5 95 0 0 0 100 0 0 95 100 195ALx-R-43 Pos 5 45 50 0 5 95 0 0 145 95 240 ALx-NR-4 Neg 25 65 10 0 0 9010 0 85 110 195 ALx-NR-7 Neg 70 30 0 0 100 0 0 0 30 0 30 ALx-NR-9 Pos 4060 0 0 0 100 0 0 60 100 160 ALx-NR-10 Neg 80 10 10 0 10 80 10 0 30 100130 ALx-NR-11 Pos 90 10 0 0 10 90 0 0 10 90 100 ALx-NR-12 Pos 10 20 70 00 100 0 0 160 100 260 ALx-NR-14 Neg 50 20 30 0 10 20 70 0 80 160 240ALx-NR-18 Pos 88 10 2 0 2 88 10 0 14 108 122 ALx-NR-20 Neg 80 20 0 0 2050 30 0 20 110 130 ALx-R-1 Neg 10 10 80 0 0 90 10 0 170 110 280 ALx-R-4Pos 20 80 0 0 0 60 40 0 80 140 220 ALx-R-5 Pos 20 40 40 0 0 60 40 0 120140 260 ALx-R-6 Neg 5 35 60 0 0 100 0 0 155 100 255 ALx-R-7 Neg 10 45 450 0 80 20 0 135 120 255 ALx-R-8 Pos 10 25 25 40 0 40 60 0 195 160 355ALx-R-9 Neg 5 35 60 0 0 100 0 0 155 100 255 ALx-R-10 Neg 5 95 0 0 0 1000 0 95 100 195 ALx-R-11 Neg 2 30 68 0 0 100 0 0 166 100 266 ALx-R-13 Pos93 5 2 0 25 75 0 0 9 75 84 ALx-R-14 Neg 10 80 10 0 0 50 50 0 100 150 250ALx-R-15 Pos 20 20 60 0 0 80 20 0 140 120 260 ALx-R-16 Pos 10 60 30 0 020 80 0 120 180 300 ALx-R-17 Neg 70 30 0 0 0 50 50 0 30 150 180 ALx-R-18Pos 5 15 80 0 0 0 100 0 175 200 375 ALx-R-19 Neg 100 0 0 0 10 20 70 0 0160 160 ALx-R-20 Pos 20 50 30 0 0 100 0 0 110 100 210 ALx-R-45 Pos 50 4010 0 10 70 20 0 60 110 170 ALx-R-44 Neg 0 50 50 0 80 20 0 0 150 20 170ALx-R-48 Neg 20 70 10 0 0 100 0 0 90 100 190 ALx-R-47 Pos 0 100 0 0 0100 0 0 100 100 200 ALx-R-54 Neg 100 0 0 0 0 100 0 0 0 100 100 ALx-R-56Neg 60 40 0 0 0 70 30 0 40 130 170 ALx-R-52 Pos 20 80 0 0 10 60 30 0 80120 200 ALx-R-57 Pos 0 100 0 0 0 100 0 0 100 100 200 ALx-R-49 Neg 95 5 00 30 70 0 0 5 70 75

TABLE 36 Histological Scoring, TL-B, Platinum/Taxane TTNT Case ID inHer2 Nuclear Cytoplasmic Nuclear Cytoplasmic Total the test set status 01 2 3 0 1 2 3 score score score Pl-1 Neg 5 95 0 0 0 80 20 0 95 120 215Pl-2 Pos 80 20 0 0 20 80 0 0 20 80 100 Pl-3 Neg 5 95 0 0 0 100 0 0 95100 195 Pl-4 Neg 10 90 0 0 0 100 0 0 90 100 190 Pl-5 Neg 60 39 1 0 0 1000 0 41 100 141 Pl-6 Pos 0 0 0 Pl-7 Neg 70 30 0 0 70 30 0 0 30 30 60 Pl-8Pos 80 20 0 0 60 40 0 0 20 40 60 Pl-9 Pos 0 0 0 Pl-10 Neg 0 50 50 0 0100 0 0 150 100 250 Pl-11 Pos 5 95 0 0 0 100 0 0 95 100 195 Pl-12 Neg 3070 0 0 30 70 0 0 70 70 140 Pl-13 Pos 30 40 30 0 0 100 0 0 100 100 200Pl-14 Pos 90 10 0 0 40 60 0 0 10 60 70 Pl-15 Neg 0 0 0 Pl-16 Neg 5 95 00 0 100 0 0 95 100 195 Pl-17 Neg 50 50 0 0 0 100 0 0 50 100 150 Pl-18Neg 0 0 0 Pl-19 Neg 0 0 0 Pl-20 Neg 10 90 0 0 70 30 0 0 90 30 120 Pl-21Pos 5 45 50 0 0 100 0 0 145 100 245 Pl-22 Pos 50 50 0 0 40 60 0 0 50 60110 Pl-23 Neg 20 80 0 0 0 80 0 0 80 80 160 Pl-24 Pos 20 70 10 0 20 40 400 90 120 210 Pl-25 Pos 0 0 0 Pl-26 Pos 60 40 0 0 0 60 40 0 40 140 180Pl-27 Neg 0 50 50 0 0 100 0 0 150 100 250 Pl-28 Pos 5 95 0 0 0 40 60 095 160 255 Pl-29 Pos 0 5 95 0 0 100 0 0 195 100 295 Pl-30 Pos 5 95 0 0 595 0 0 95 95 190 Pl-31 Pos 5 85 10 0 0 100 0 0 105 100 205 Pl-32 Neg 5050 0 0 0 70 30 0 50 130 180 Pl-33 Neg 40 60 0 0 0 100 0 0 60 100 160Pl-34 Pos 100 0 0 0 100 0 0 0 0 0 0 Pl-35 Pos 10 85 5 0 0 100 0 0 95 100195 Pl-36 Pos 30 60 10 0 20 75 5 0 80 85 165 Pl-37 Neg 20 20 60 0 100 00 0 140 0 140 Pl-38 Neg 50 40 10 0 0 100 0 0 60 100 160 Pl-39 Pos 30 700 0 20 70 10 0 70 90 160 Pl-40 Neg 60 40 0 0 0 100 0 0 40 100 140 Pl-41Neg 20 80 0 0 0 50 50 0 80 150 230

With two libraries in hand that reciprocally show preferential stainingfor either B or NB cases, we tested use fo PHC scores for thedifferentiation and prediction of responders and non-responders totrastuzumab-based regimens in the 45 independent test cases. See FIG.17A. We assessed the PHC scores of TL-B and TL-NB by receiver operatingcharacteristic (ROC) curves, and calculated area under the curve (AUC)values (FIGS. 17Mi-ii). For TL-NB, an AUC value of 0.703 was obtainedbased on the scoring of the nucleus (22) (FIG. 17Mi), whereas TL-Byielded an AUC value of 0.688 (FIG. 17Mii) based on the scoring of bothnucleus and cytoplasm. These AUC values from the individual librariesindicate that TL-NB and TL-B provide reliable information thatdistinguishes the NB and B phenotypes. To predict the patient's responseto trastuzumab-based regimens with a multivariate method that uses PHCscores from both TL-NB and TL-B staining, a logistic regression modelwas developed. Specifically, the binary outcome of benefit- ornon-benefit status was used as the dependent variable; the stainingscores of TL-NB and TL-B, respectively, were treated as independentvariables. We then assessed the performance of the model by ROC curveanalysis. As a result, by combining the data from the two libraries, theAUC-value increased to 0.804, indicating improved performance due to thereciprocal nature of the training schemes (FIG. 17Miii, indicated byAUC=0.804). The statistical reliability of this analysis was furtherverified by 10-fold cross validation (CV), which resulted in an AUCvalue of 0.760 (FIG. 17Miii, indicated by AUC=0.760). We then comparedthese PHC-based NB and B classifications with those predicted by HER2immunohistochemistry scoring of the same 45 cases test set (FIG. 17Miii,indicated by AUC=0.410). The HER2 IHC results yielded an AUC value of0.410, indicating that TL-NB and TL-B outperformed conventional HER2 IHCin classifying trastuzumab B and NB cases in this cohort.

TL-NB and TL-B were able to effectively classify B and NB patients thathad low levels of HER2-expression by IHC with an AUC value of 0.8. FIG.17N shows the receiver operating characteristic (ROC) curves fordifferentiation between patients, benefiting and not fromtrastuzumab-based regimens, using combined histological scores fromlibraries TL NB and TL B polyligand histochemistry (PHC) staining in thetest set of 45 cases, shown separately in the group of HER2+ cases (A)and in the group of HER2−/low cases (B). Hence, the ability of thelibraries to differentiate NB from B-cases does not depend solely on theHER2 status.

To determine whether TL-NB and TL-B were revealing information aboutresponse to trastuzumab-based regimens and not only classifying patientswith a favorable prognosis regardless of trastuzumab treatment, westained FFPE tumor tissues from an independent cohort of 33 patients whowere treated with a platinum/taxane combination without trastuzumab.Like all other samples in this study, the samples from thisnon-trastuzumab cohort were collected prior to treatment. The AUC of thecombined libraries on 33 HER2-cases independent from the test set whoreceived platinum/taxane combination therapy instead of trastuzumab was0.302 (FIG. 17Miii, indicated by AUC=0.302), indicating that theperformance of TL-NB and TL-B relate to the molecular profiledetermining the response to the presence of trastuzumab in the treatmentregimens. Moreover, trastuzumab has been reported to be substantiallyless effective in estrogen receptor (ER)-positive breast cancer (23,24), but we found that the ER status of all cases enrolled in our studyshowed no correlation with the benefit from trastuzumab-based therapy.See FIG. 17O, which shows that in the tested population, benefit fromtrastuzumab does not correlate with hormonal status. ER status for thepatients in this Example is shown in Tables 31-32. Taken together, thesedata indicate that the application of these libraries to the 45-casestest-set classifies patients with benefit from patients without benefitfrom trastuzumab-containing treatment with high accuracy, regardless oftheir HER2-status.

We next sought to evaluate the ability of PHC to select the fraction ofpatients that benefit from trastuzumab-based regimens for more than 180days. FIG. 17Pi-ii show Kaplan-Meier curves of trastuzumab-treatedbreast cancer patients stratified by polyligand profiling. In FIG. 17Pi,the shortest distance between the ROC curves to point (specificity andsensitivity=100%) determines the cutoff of test positive and negative,and is represented as dot on the line indicated by AUC=0.804 in FIG.17Miii (Sensitivity: 66.6%; Specificity: 86.7%). An “event” was definedas the time point (days) at which a patient either deceased from canceror at which trastuzumab-based treatment changed. Median time of benefitis 604 days for patients tested positive ((FIG. 17Pi, upper curve),n=22, event=11) and 129 days for patients tested negative ((FIG. 17Pi,lower curve), n=23, event=18). HR=0.320, 95% CI: 0.146-0.703; log-rankp=0.003. The small vertical lines mark cases that were censored due toabsence of treatment follow-up data. See Table 32. FIG. 17Pii shows theKaplan-Meier curve of trastuzumab-treated breast cancer patientsstratified by tumors' HER2 status. HER2 status of patients with bothHER2 IHC and HER2 ISH test results was determined according to thefollowing rules. 1. HER2 positive, if any test shows positive result;else 2, HER2−/low, if any test shows negative result; else 3, NA, ifboth test show equivalent result. Median time of benefit was 250 daysfor HER2 positive cases ((FIG. 17Pii, lower curve, n=25, event=16), and369 days for HER2−/low cases ((FIG. 17Pii, upper curve; n=18, event=12).HR=1.39, 95% CI: 0.62-3.13; log-rank p=0.418. Accordingly, the resultingKaplan-Meier curves show that cases with positive test results exhibiteda significantly longer TTNT (FIG. 17Pi, upper curve) than those testednegative (FIG. 17Pi, lower curve). The median event-free time, i.e. thetime that elapsed before the treatment regimens changed, increased from129 days for the test-negative to 604 days for the test-positive cases.For comparison, we analyzed the prognostic value based on the tumors'HER2 status as determined by IHC. In this case, the KM-curves did notreveal any significant difference in TTNT between patients that wereHER2+ or Her2−/low (FIG. 17Pii). These data indicate that polyligandprofiling can be employed to generate a predictive assay thatdifferentiates between patients that benefit from a certain treatmentregimens from those that do not.

In this Example, we demonstrated a prototypical polyligand profilingapproach with potential to improve patient stratification for cancertherapy. The approach was applied to train the libraries TL-B and TL-NBfor classifying trastuzumab response by TTNT. Polyligand profiling isnot limited to this condition and will be applicable for differentiatinga wide variety of phenotypes depending on the question at hand and theavailability of suitable samples. Previous studies have usedmorphology-based enrichment of ssODN libraries on cancer tissues (seeherein, references 25, 26) in order to identify new biomarkers. In thisstudy, to account for the heterogeneity of molecular composition and thecomplex interactomes that reflect intra- and inter-tumoral variabilityit is reasonable that library training as introduced here enriches for acomplex variety of ligands.

Without optimization, the PHC-test developed in this Exampleoutperformed the standard HER2 IHC in differentiating the patients inthis cohort who benefit from trastuzumab-based treatment from those thatdo not. See FIGS. 17M, P and related discussion above. These dataindicate that the PHC-test can provide a CDx, in this case one that aimsat identifying the 50-70% of HER2+ patients who will not benefit fromtrastuzumab. Conversely, it has been reported that 16-45% of patientswith breast cancer who express low levels of Her2 derived benefit fromadjuvant trastuzumab in combination chemotherapy (27-29). The data shownin FIGS. 17M, P indicate that a CDx based on polyligand profiling alsohas the potential to identify the patients who benefit fromtrastuzumab-containing regimens from the group expressing low levels ofHer2.

Polyligand profiling allows accessing the molecular information that isreflected by the perturbations within the vast diversity of biologicalcomplexes that constitute cellular interactomes within or among tumors.In addition to predicting patients with benefit or without benefit froma given drug treatment regimens, the PHC approach can advance precisiononcology in additional ways. For example, when applied to existingtherapies as well as to new compounds in clinical trials polyligandprofiling could reduce the administration of toxic therapies that areineffective, and thereby could increase the success rate of new drugs.

References cited in this Example, all incorporated by reference hereinin their entirety.

-   1. D. Hanahan, R. A. Weinberg, Hallmarks of cancer: the next    generation. Cell 144, 646-674 (2011).-   2. M. Gerlinger et al., Intratumor heterogeneity and branched    evolution revealed by multiregion sequencing. N Engl J Med 366,    883-892 (2012).-   3. L. A. Garraway, E. S. Lander, Lessons from the cancer genome.    Cell 153, 17-37 (2013).-   4. V. Prasad, Perspective: The precision-oncology illusion. Nature    537, S63 (2016).-   5. V. Prasad, T. Fojo, M. Brada, Precision oncology: origins,    optimism, and potential. Lancet Oncol 17, e81-86 (2016).-   6. G. L. Klement et al., Future paradigms for precision oncology.    Oncotarget 7, 46813-46831 (2016).-   7. R. Kurzrock, F. J. Giles, Precision oncology for patients with    advanced cancer: the challenges of malignant snowflakes. Cell Cycle    14, 2219-2221 (2015).-   8. J. Shrager, J. M. Tenenbaum, Rapid learning for precision    oncology. Nat Rev Clin Oncol 11, 109-118 (2014).-   9. M. J. Duffy, J. Crown, Companion biomarkers: paving the pathway    to personalized treatment for cancer. Clin Chem 59, 1447-1456    (2013).-   10. M. P. Stumpf et al., Estimating the size of the human    interactome. Proc Natl Acad Sci USA 105, 6959-6964 (2008).-   11. Z. Mitri, T. Constantine, R. O'Regan, The HER2 Receptor in    Breast Cancer: Pathophysiology, Clinical Use, and New Advances in    Therapy. Chemother Res Pract 2012, 743193 (2012).-   12. H. J. Burstein, The distinctive nature of HER2-positive breast    cancers. N Engl J Med 353, 1652-1654 (2005).-   13. R. Bartsch, C. Wenzel, G. G. Steger, Trastuzumab in the    management of early and advanced stage breast cancer. Biologics 1,    19-31 (2007).-   14. I. Petak, R. Schwab, L. Orfi, L. Kopper, G. Keri, Integrating    molecular diagnostics into anticancer drug discovery. Nat Rev Drug    Discov 9, 523-535 (2010).-   15. D. J. Slamon et al., Human breast cancer: correlation of relapse    and survival with amplification of the HER-2/neu oncogene. Science    235, 177-182 (1987).-   16. J. Horton, Trastuzumab use in breast cancer: clinical issues.    Cancer Control 9, 499-507 (2002).-   17. M. Famulok, J. S. Hartig, G. Mayer, Functional aptamers and    aptazymes in biotechnology, diagnostics, and therapy. Chem. Rev.    107, 3715-3743 (2007).-   18. M. Famulok, G. Mayer, Aptamer modules as sensors and detectors.    Acc Chem Res 44, 1349-1358 (2011).-   19. TTNT is a FDA-approved clinical endpoint that is robustly    captured by electronic medical record systems and therefore is    generally available.-   20. U.S. Department of Health and Human Services Food and Drug    Administration Center for Drug Evaluation and Research (CDER),    Guidance for industry: clinical trial endpoints for the approval of    cancer drugs and biologics.-   21. Z. Gatalica, S. M. Lele, B. A. Rampy, B. A. Norris, The    expression of Fhit protein is related inversely to disease    progression in patients with breast carcinoma. Cancer 88, 1378-1383    (2000).-   22. TL-NB staining of the cytoplasm was not informative for the    differentiation of B and NB cases.-   23. L. Lousberg, J. Collignon, G. Jerusalem, Resistance to therapy    in estrogen receptor positive and human epidermal growth factor 2    positive breast cancers: progress with latest therapeutic    strategies. Ther Adv Med Oncol 8, 429-449 (2016).-   24. S. Loi et al., Effects of Estrogen Receptor and Human Epidermal    Growth Factor Receptor-2 Levels on the Efficacy of Trastuzumab: A    Secondary Analysis of the HERA Trial. JAMA Oncol 2, 1040-1047    (2016).-   25. H. Wang et al., Morph-X-Select: Morphology-based tissue aptamer    selection for ovarian cancer biomarker discovery. Biotechniques 61,    249-259 (2016).-   26. S. Li et al., Identification of an aptamer targeting hnRNP A1 by    tissue slide-based SELEX. J Pathol 218, 327-336 (2009).-   27. S. Paik, C. Kim, N. Wolmark, HER2 status and benefit from    adjuvant trastuzumab in breast cancer. N Engl J Med 358, 1409-1411    (2008).-   28. S. Ithimakin et al., HER2 drives luminal breast cancer stem    cells in the absence of HER2 amplification: implications for    efficacy of adjuvant trastuzumab. Cancer Res 73, 1635-1646 (2013).-   29.    clinicaltrials.gov/ct2/show/NCT01275677?term=NSABP+B01275647&rank=01275671.

Example 22 Polyligand Profiling Materials and Methods

This Example provides Materials and Methods used in Example 21 above.

FFPE Tissue Cases

The study was performed with Western Institutional Review Boardapproval, 45 CFR 46.101(b)(4).

This study included cases from women with invasive breast cancer thatreceived trastuzumab-based treatment first line after tissue collectionwith a sufficient number of properly fixed and embedded slidesavailable. Cases with in situ cancer, improperly fixed or crushed tissuesections were not included in this study. Cases with incomplete staining(i.e. insufficient coverage of the tissue with binding solution) andother technical problems with the assay performance were excluded fromanalysis.

Serial tissue sections of 51 patients with breast cancer that hadtime-to-next treatment (TTNT) data from trastuzumab therapy were usedfor training of the ssODN libraries and testing of TL-NB and TL-B. Aseparate group of 33 cases that had TTNT data from platinum-taxanetreatment was used as a control set to evaluate the specificities ofTL-NB and TL-B towards trastuzumab-based TTNT. Excised tissue containingboth tumor and normal parts, was formalin-fixed, paraffin-embedded andserially sectioned (4 μm). Haematoxylin and eosin (H&E) staining wasperformed for 1-2 slides of each case and served for initialpathological diagnosis. Pre-treatment of the FFPE tissue slides beforethe enrichment included incubating slides at 60° C. for 1.5 h, followedby automated deparaffinization and epitope retrieval on the VentanaUltraView Autostainer (Ventana Medical Systems, Inc., Tuscon, Ariz.).Specifically, deparaffinization at 72° C. for 24 min, dehydration byethanol, epitope retrieval at 90° C. for 36 min and 100° C. for 4 min(pH 8), followed by peroxidase inhibition (H₂O₂, 1%≤x<5%) and washingslides with detergent (Dawn 1-00; P&G Professional, The Procter & GambleCompany, Cincinnati, Ohio) to remove residual liquid coverslip. Fortesting of the enriched libraries, deparaffinization was performedmanually, by incubation at 60° C. for 1.5 hour, followed by epitoperetrieval on PT-Linker (Dako, Agilent Technologies, Santa Clara, Calif.)at 97° C. for 20 min, pH 9.

Treatment regimens and pathological diagnoses for 84 cases can be foundin Tables 29-32. A subset of 3 non-benefiting (NB) breast cancer casesand 3 benefiting (B) breast cancer cases was selected for enrichment.Patients who stayed on their initially prescribed trastuzumab therapyfor at least 181 days were classified as B; patients whose treatmentchanged within 180 days were classified as NB. Clinical information forthe training cases can be found in Tables 29-30. For enrichmentpurposes, tissue areas with breast carcinoma were utilized as positiveselection targets, while adjacent non-malignant tissue as well ascarcinoma tissue from patients with alternative response was used ascounter selection targets.

ssDNA Library Design and Reproduction with Unequal Length PrimersAsymmetrical PCR

The random ssDNA library (naïve F-TRin-35n-B 8-3s library) contains 35random nucleotides flanked by constant regions. Specifically, the naïvelibrary comprises a 5′ region (5′ CTAGCATGACTGCAGTACGT (SEQ ID NO. 4))followed by a random naïve oligonucleotide sequence of 35 nucleotidesand a 3′ region (5′ CTGTCTCTTATACACATCTGACGCTGCCGACGA (SEQ ID NO. 5)).This library was synthesized at Integrated DNA Technologies (IDT,Coralville, Iowa, USA), pooled in equimolar amounts and PCR amplified toadd biotin to the 5′-end. The library constant regions are complimentaryto primers: reverse: 5′-Biosg-CTAGCATGACTGCAGTACGT-3′ (SEQ ID NO. 4) andforward: 5′-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO.203062)/iSp9//iSp9/TCGTCGGCAGCGTCA-3′ (SEQ ID NO. 203063)), which wereused in asymmetric PCR to generate majority of the target strand. Theinternal spacers iSp9 (Internal triethylene glycol Spacer, IDT) of theforward primer prevented extension of the complimentary strand, whilethe addition of poly-A tail resulted in longer length of forwardstrands, allowing for size separation and target strand ssDNA recoveryfollowing gel excision from 4% denaturing agarose E-gels with finalpurification by gel extraction column (Nucleospin, MACHEREY-NAGEL GmbH &Co. KG, Diiren, Del.). Biotinylated antisense library was used forenrichment. Asymmetric PCR mixture (100 μl) contained 5× Q5 PCR buffer,0.2 mM dNTPs, 0.08 μM of forward primer, 30 μM of reverse primer, 0.01pmol template (of pure library) or 57 μl of post-dissection solutioncontaining library/tissue (after enrichment) and 2U of Q5 Hot StartHigh-Fidelity DNA polymerase (New England Biolabs, Ipswich, Mass.). PCRprogram included initial denaturation at 98° C. for 30 sec, followed bycycle of denaturation, annealing (60° C. for 30 sec) and extension (72°C. for 3 min), and final extension was at 72° C. for 5 min. For purelibrary 15 cycles of amplification were performed, for libraries duringenrichment number of cycles varies between 15 and 30 depends on therecovery. Asymmetric PCR products were mixed with denaturing buffer (180mM NaOH, 6 mM EDTA), heated at 70° C. for 10 min, cooled down on ice for3 min, loaded ˜20 μl on 4% agarose SYBRGold gel (E-GEL EX Gels, G401004,Life Technologies), separated for 15 min. Single stranded reverse strandwas cut, gel pieces were combined with NTC buffer (Nucleospin,Macherey-Nagel), melted at 50° C. for 5-10 min until all pieces gotmolten. 700 μl of melted agarose was loaded onto Nucleospin column andthen followed standard procedure for ssDNA purification. Purified DNAwas eluted with 30 μl of NE buffer.

FFPE Tissue Slide-Based SELEX

Enrichment of ssODNs libraries toward Trastuzumab response was performedaccording to the scheme in FIG. 17C. Treatment regimens for enrichmentcases can be found in Table 29-30. In the enrichment of each library,first three rounds were performed on positive cases only, followed byadditional three rounds with two counter selections cases and onepositive case. For positive selection, 400 μl of blocking buffer (0.8ng/ul Salmon DNA (Life Technologies, Thermo Fisher Scientific Inc.,Waltham, Mass., USA), 0.8 ng/μl tRNA (Life Technologies), 1 μg/μl HSA(Sigma), 0.5% F127 (Thermo Fisher) and 3 mM MgCl₂ in 1×PBS) was mixedwith 90 μl of ssODN library solution (7 pmol for round 1, 3.5 pmol forfollowing rounds in 1×PBS, 3 mM MgCl₂) on top of the Agilent gasketslide (Agilent Technologies, Santa Clara, Calif.). FFPE tissue slide,after deparaffinization and epitope retrieval, was mounted on top of thegasket slide containing binding cocktail and incubated for 1 hour inAgilent microarray hybridization chambers with rotation at RT. Afterincubation, slides were washed by dipping into 2×750 ml washing buffer(1×PBS, 3 mM MgCl₂) buffer, 3 dips into each jar. Next, 490 μl ofnuclear fast red (NFR), supplemented with 3 mM MgCl₂, was added to theslide for 45 s and washed by 6 times dipping in 750 ml washing buffer.Based on the initial pathological diagnosis from corresponding H&Eslides, cancer areas were dissected and transferred into 180 μl water,which served as a template for asymmetric PCR with unequal lengthprimers to generate single stranded library for next round (see protocolabove). Remaining normal tissue served for internal counter selection.This protocol was repeated for 3 rounds. For negative selection, bindingcocktail was added directly to the tissue of counter selection slidesand incubated for 1 hour in humidity chamber. After incubation, maximumvolume of supernatant was collected. Additionally, 50 μl of blockingbuffer was applied to collect the unbound ssODNs. Combined supernatantwas added to the 2^(nd) counter selection slide and incubated for 1hour. After incubation supernatant was collected the same way as beforeand applied to the slide from positive case for another hour incubation,done this time in the Agilent microarray hybridization chamber. Thefollowing steps, washing, staining and PCR, were the same as describedabove.

Polyligand-Histochemistry (PHC) Screening of the Enriched Libraries

Staining of FFPE tissue slides with enriched libraries was performed onDako Autostainer. After baking slides at 60° C. for 1.5 hour, epitoperetrieval was done on Dako PT-Linker at pH9, 98° C., 22 min. Thestaining on Dako Autostainer includes 5 min peroxidase inhibition with450 μl of solution, containing disodium hydrogenorthophosphate 5%<=x<7%,H₂O₂ 3%<=x<5%, phosphoric acid, monosodium salt, monohydrate 1%<=x<2%, 1hour incubation with 450 ul of binding cocktail (3.5 pmol of enrichedlibrary, 0.65 ng/μl Salmon DNA, 0.65 ng/μl tRNA, 10% BlockAid (LifeTechnologies), 30 min incubation with 450 μl of Streptavidin Poly-HRP,supplemented with 3 mM MgCl₂, 10 min staining with DAB solution,supplemented with 3 mM MgCl₂, followed by 5 min incubation with 450 μlof Hematoxylin (2 ng/μl final conc.). Rinsing with 1×PBS, 3 mM MgCl₂buffer was done between each step. Finally, the stained slides weredehydrated with ethanol, xylene and covered by coverslip for long-termstorage. Microscopy was done on Olympus BX41 (Olympus Corporation of theAmericas, Center Valley, Pa., USA).

Histological scores for both nuclear and cytoplasmic staining werecalculated as sum between intensity levels (1, 2 and 3) multiplied bythe percentage of the cells with this particular intensity.

Statistical Analysis

Firstly, the ability of each single library to classify Trastuzumabresponders and non-responders was assessed by ROC curves and AUCs. See,e.g., FIGS. 17J-K. To predict the patient's response to trastuzumabtherapy with a multivariate method using PHC scores from both libraryTL-NB and TL-B staining, a logistic regression model was developed.Specifically, a binary outcome of responder/non-responder was used asthe response variable, and staining scores of libraries TL-NB and TL-Bwere treated as independent variables (FIG. 17J (C), solid line labeledAUC=0.804). A 10-fold cross-validation was conducted to assess thegeneralizability of the model's prediction performance, in which thedata set was randomly split into 10 equal parts exclusively. A logisticregression model was built on 9 parts, and subsequently tested on the 1hold-out part. This process was iterated throughout the 10 parts, andonly the predicted probability was collected for further assessment(FIG. 17J (C), dashed line labeled AUC=0.760).

The end points of time to next treatment (TTNT) were defined as eitherthe time of next non-trastuzumab treatment or death. Patients withoutthe next non-trastuzumab treatment or death information were censored atthe last contact date (see vertical marks in the Kaplan-Meier curves(FIG. 17M). A Cox-PH model was fitted using either tumors' HER2 statusor PHC test results as the independent variable. Median survival timewas calculated from the Kaplan-Meier estimate. The Log-rank test wasperformed to evaluate the significance of TTNT survival between groups.All analysis was conducted using the “survival” r package.

Example 23 Alternate Oligonucleotide Probe Assay Classifies Outcomes ofBreast Cancer Patients Treated with Trastuzumab-Based Therapy

Background: Although trastuzumab is a highly effective targeted therapy,approximately 50% of patients with HER2+ breast cancers do not benefitfrom trastuzumab-based regimens. We developed a poly-ligandaptamer-based approach that outperforms traditional HER2 testing in itsability to classify patients likely to derive benefit from trastuzumab.

Methods: A library of 10¹³ biotinylated ssDNA oligodeoxynucleotides(ssODN) was incubated on FFPE tissue slides of patients who eitherbenefitted (B) or did not benefit (NB) from trastuzumab-based therapiesbased on time to next treatment. Unbound ssODNs were discarded and boundssODNs were retained for two subsequent rounds of positive selection.Starting at round 4, the partially enriched library was applied to casesrepresenting the alternate phenotype (NB for B and vice versa) fornegative selection and supernatant containing unbound ssODNs wascollected and retained. Libraries obtained after two more rounds ofnegative selection were used as probes to stain and score 30 B and 15 NBindependent cases by Polyligand Histochemistry (PHC). The ability of thetrained libraries to classify patients who benefitted fromtrastuzumab-based treatment was assessed by calculating AUC values fromROC curves. Kaplan-Meier (KM) plots were used to compare outcomes withtest results. [001117] Results: Two libraries effectively classified Band NB groups (ROC AUC of 0.8). In contrast, standard HER2 IHC yieldedan AUC of 0.4. Median duration of trastuzumab treatment was 604 days fortest-positive patients and 129 days for test-negative patients(HR=0.320, 95% CI:0.146-0.703; log-rank p=0.003), indicating that PHCscoring was highly effective for classifying distinct clinical outcomes.By HER2 status alone (IHC), median duration of trastuzumab treatment of250 days for HER2+ cases and 369 days for HER2−/low cases (HR=1.39, 95%CI: 0.62-3.13; log-rank p=0.418).

Conclusions: These results indicate that polyligand profiling provides ahigh-performance systems biology approach to generating predictiveassays that can be used to identify patients more or less likely tobenefit from targeted therapies such as trastuzumab.

Example 24 Selection of Aptamers on FFPE Tissue from IndividualsDiagnosed with Colorectal Cancer and Treated with FOLFOX and Bevacizumab

Bevacizumab is a recombinant humanized monoclonal antibody that blocksangiogenesis by inhibiting vascular endothelial growth factor A(VEGF-A). VEGF-A is a chemical signal that stimulates angiogenesis in avariety of diseases, especially in cancer. Bevacizumab, sold under thetrade name Avastin, was approved by the FDA in February 2004 for use inmetastatic colorectal cancer (CRC) when used with standard chemotherapytreatment (as first-line treatment) and with 5-fluorouracil-basedtherapy for second-line metastatic colorectal cancer. FOLFOX is achemotherapy regimen for treatment of colorectal cancer, made up ofthree drugs: 1) FOL—Folinic acid (leucovorin); 2) F—Fluorouracil (5-FU);and 3) OX—Oxaliplatin (Eloxatin). FOLFOX and bevacizumab may be used asfirst-line treatment for metastatic CRC.

Currently, there is no good way to predict efficacy of FOLFOX withBevacizumab as a treatment in cancer therapy. The goal of this Examplewas the development of oligonucleotide probe libraries that can be usedin an assay that predicts the response to a combinational therapy withFOLFOX/bevacizumab in individuals diagnosed with colorectal cancer. Theapproach taken is similar to Examples 19-20 above. Briefly, theselection of oligonucleotide probes was performed on FFPE CRC tissuesamples. Patients were divided into two cohorts based on the time theyhave stayed on FOLFOX (TNT; time to next treatment): responder andnon-responder. A non-responder stayed on FOLFOX for a maximum of 124days while a responder was on this treatment for at least 241 days.Bevacizumab treatment varied from patient to patient. It might havestarted at the same time as FOLFOX or later and ended at the same time,earlier or later than FOLFOX. An enrichment scheme is outlined in FIG.18A with positive and negative selection steps illustrated in FIG. 18Band FIG. 18C, respectively.

Six rounds of positive selection of 7 enrichments for responder and 4enrichments for non-responders were performed, creating 11 pools ofenriched oligonucleotide probes. Counter selection was performed forresponder cases on the non-responder samples and vice versa. Anexemplary set of slides is shown in FIGS. 18D-E, which shows staining ofan FFPE sample used for positive selection in enrichment “M” stainedwith H&E (FIG. 18D) or the probe library M (FIG. 18E). The figure showsstaining for cancer tissue; tumor site: colon; specimen: colon. Theslides are shown at 20× magnification. Table 37 shows results ofstaining intensity of the indicated tissue samples with the indicatedprobe library.

TABLE 37 Enriched oligonucleotide probes Enriched Specimen used for Non-Probe Library positive selection Cancer Cancer Non- A Rectum ++ −responder C Colon +++ No data E +++ No data F ++ + Responder G ++ ++ H++ − I +++ +++ J +++ + K ++ ++ L ++ + M +++ +

To test the enriched libraries, one non-responder case and 5 respondercases that were not part of the enrichment were stained with all 11libraries. In addition, the cases used for positive selection werestained with the corresponding libraries. Exemplary results for stainingof one test sample, Sample 2, are shown in FIGS. 18F-G. The samplecharacteristics include: Responder; tumor site: descending colon;specimen: descending colon. FIG. 18F shows staining of a cancer samplefrom the Sample 2 slide. The library (A-M) are indicated over each slidein the figure. As shown, slightly darker staining was observed in slidesH-M as compared to the others. FIG. 18G shows staining of non-cancertissue from the Sample 2 slide. In this setting, staining was lighterand of similar intensity across all samples.

Another set of enrichment experiments was performed on a fixed samplecomprising tumor tissue from an individual considered to be a responderto bevacizumab. For positive selection, the oligonucleotide probelibrary was binding to the entire tissue on the slide but only thecancer tissue was scraped off the slide, then the library was recoveredfrom the scraped tissue and amplified. After three such rounds, threerounds of two steps of negative selection (tissue from two individualsnot responding to Avastin) and one positive selection as before wereperformed. Round 7 included one more positive selection. For round 8,the enrichment strategy was altered. Instead of doing enrichment oftissue fixed to slides, the cancer tissue and the non-cancer tissue werescraped and separated in different tubes. To perform enrichments intubes, we used centrifugation at 100,000×g to separate the tissue fromunbound library. Four rounds of enrichment on scraped cancer tissue withcounter selection on scraped non-cancer tissue were performed. Thelibrary after 12 rounds was used for probing and selection of aptamerthat bound stronger to cancer relative to non-cancer tissue. Foursequences recovered from the enrichment are shown in Table 38. The tableindicates the variable region of the identified sequences along with thefull length sequence with 5′ region CTAGCATGACTGCAGTACGT (SEQ ID NO 4)and a 3′ region CTGTCTCTTATACACATCTGACGCTGCCGACGA (SEQ ID NO 5)surrounding the variable region. These sequences can be used toidentify, e.g., by staining, cancer tissue from a responder tobevacizumab. In the table, the sequences with name comprising “RC” arethe reverse complement sequences used as non-binding controls.

TABLE 38 Oligonucleotide probes that bind bevacizumab respondercancer tissue Variable Region SEQ ID SEQ ID (5′->3′) NO Seq nameFull sequence (5′->3′) NO GCTTCCCGC 203064 5825D-/5Biosg/CTAGCATGACTGCAGTACGTGC 203068 AACCGTCCT R12c-S1-TTCCCGCAACCGTCCTACCCATCCTACGCC ACCCATCCT 5′biotinTCCCTGTCTCTTATACACATCTGACGCTGC ACGCCTCC CGACGA 5825D-/5Biosg/TCGTCGGCAGCGTCAGATGTGT 203069 R12c-S1RC-ATAAGAGACAGGGAGGCGTAGGATGGGTAG 5′biotin GACGGTTGCGGGAAGCACGTACTGCAGTCATGCTAG TGGTCACCG 203065 5825D- /5Biosg/CTAGCATGACTGCAGTACGTTG 203070CTTCCGCTG R12c-S2- GTCACCGCTTCCGCTGGTACGCCCCACCCC GTACGCCCC 5′biotinATAACTGTCTCTTATACACATCTGACGCTG ACCCCATAA CCGACGA 5825D-/5Biosg/TCGTCGGCAGCGTCAGATGTGT 203071 R12c-S2RC-ATAAGAGACAGTTATGGGGTGGGGCGTACC 5′biotin AGCGGAAGCGGTGACCAACGTACTGCAGTCATGCTAG ACCTAGCTG 203066 5825D- /5Biosg/CTAGCATGACTGCAGTACGTAC 203072CTCCCTACT R12c-S3- CTAGCTGCTCCCTACTCCCTTCTTATGTAC CCCTTCTTA 5′biotinATTACTGTCTCTTATACACATCTGACGCTG TGTACATTA CCGACGA 5825D-/5Biosg/TCGTCGGCAGCGTCAGATGTGT 203073 R12c-S3RC-ATAAGAGACAGTAATGTACATAAGAAGGGA 5′biotin GTAGGGAGCAGCTAGGTACGTACTGCAGTCATGCTAG TTCATGCTG 203067 5825D- /5Biosg/CTAGCATGACTGCAGTACGTTT 203074TTCCATGAC R12c-S4- CATGCTGTTCCATGACCTCCACATTTCTCA CTCCACATT 5′biotinCGCTGACTGTCTCTTATACACATCTGACGC TCTCACGCT TGCCGACGA GA 5825D-/5Biosg/TCGTCGGCAGCGTCAGATGTGT 203075 R12c-S4RC-ATAAGAGACAGTCAGCGTGAGAAATGTGGA 5′biotin GGTCATGGAACAGCATGAAACGTACTGCAGTCATGCTAG

Additional sequences were determined after round 9 above. Tissue fromresponder cancer (i.e., cancer tissue from responder patients),responder non-cancer (i.e., non-cancer tissue from responder patients),non-responder cancer (i.e., cancer tissue from non-responder patients)and non-responder non-cancer (i.e., non-cancer tissue from non-responderpatients) were scraped and bound oligonucleotides were sequenced. Thefold changes between the non-responder cancer and other tissues werecalculated after normalization of read counts from the sequencing to thetissue sizes. Table 39 shows fold changes for the non-responder cancerover the other three tissue types as indicated. The table indicates thevariable region of the identified sequences. The full length sequencescomprised 5′ region CTAGCATGACTGCAGTACGT (SEQ ID NO 4) and a 3′ regionCTGTCTCTTATACACATCTGACGCTGCCGACGA (SEQ ID NO 5) surrounding the variableregion. Any appropriate flanking sequences can be used. In Table 40, thecomplete sequences for the first six variable regions in Table 39 alongwith the complete sequences with flanking regions. These sequences canbe used to identify, e.g., by staining, cancer tissue from a responderto bevacizumab. The variables regions of additional sequences with foldchanges of at least 100% in one comparison are listed in SEQ ID NOs.203114-206478. In Table 40, the sequences with name comprising “RC” arethe reverse complement sequences and can be used as non-bindingcontrols. The sequences in Table 40 are further shown labeled withdigoxigenin (DIG). Anti-digoxigenin antibodies with high affinities andspecificity can be used in a variety of biological immuno-assays, e.g.,to visualize the oligonucleotide staining as described herein. Bothbiotin and digoxigenin labeling can be used in such applications, inaddition to any other such useful labeling systems. Any desired numberor combination of the sequences in Table 39 can be used to createoligonucleotide probes such as in Table 40.

TABLE 39Sequences with fold-change differences between bevacizumab non-respondersand responders Sample Type/Fold changes SEQ ID Responder, Non-responder,Responder, Variable Sequence 5′->3′ NO Cancer non-cancer non-cancerATATGCGATGCTAGCTCGAAGCGTGTGCAGTCCCT 203076 2 4 2GTCCAGCTCGCAATTACACTAGTTTGCCAGTAAAAG 203077 3 4 3TCCGGAGTCCATAAGACTACGGATAGCTTTGACCG 203078 3 3 9CATGCACATCGCGTTTCGGAAACAAAAGTGTAGATA 203079 2 3 4CCACACGACGTGTGACCTCGGATTCCTCATACACT 203080 2 3 3ACTGGGTACTTGAATCATTGTCGCATTTCCCTATT 203081 4 3 11CGTAGTTAAGACGCCTCAAGACACATACGGCTTGAA 203082 0 49 0TCATGACCGAGTAACGACCGACCCACATATCCGC 203083 1 37 2GTTGGCTCCCTCTAGCATATCCCCACTAGCTGG 203084 1 26 1CGCCCACTCTTGTTGTTACCGTGCTGTCCTCGGTC 203085 1 22 1GCATGTGTGGTTTCCCCAGCGCACTCGAGACGACT 203086 0 22 1GAAAAGTCAGACTTTTGAAGGATTGTGCTGGAACT 203087 1 20 1GAACATGAAGCATCGAAAGCACTTCACGTAGATTCGA 203088 1 17 0TTGTGGATTCAAGCGGTCACCACTATGCCAACTTCCA 203089 1 14 1CCACCTTTTCCACCCATTCCAGGATAGTCGCCCACGA 203090 0 13 0GTTCGGATGCCCACAGCCCGTTAAGGTATTTTCACGA 203091 0 11 0TTGTGGGGGACGTATGAGCAGATCCATGCGCGCCA 203092 0 9 1AGATCTCCGGTACGCCACCGGCGATACCGATTCAG 203093 1 9 1GTAACCCGATTTTCACGGCCGACCGCATACGCGCC 203094 0 8 1GCTAGACACTTACAGTGACGTTACTGTCTTGAGCT 203095 0 8 1GGACATTCAGGTGTCGCTCATCCAAAGCTACGTGC 203096 1 7 6GTTATCGCACTGAAAAGTAATTCCTTGTATACTCT 203097 1 7 1GCAAAGGATCCTGGTTGGGCACTTTGTTCCTTTCC 203098 0 7 0ACCCACTAGGAGGTCGTTAAAGGATAGTTGTAAACTCA 203099 2 7 2TCTTGCCTCTGTATGAGCGAGAACTTCTCGCGCGTTCA 203100 1 7 1GTCAGTCCCTGGAGTCCATGTGCTTTACCGCCGTC 203101 0 7 0GAAAGACAACACGTATACATCCCTGAGCCACCCACGA 203102 0 6 0CCATTGCTCTTACACCGCCTGTCCTTACGGTTGTGGA 203103 1 6 4TGTTTGCCGATCTCTCATCTGGAGGAACGGGGCA 203104 0 6 1GTAGTATGTTCTCTTTACTGCGACATGAGTTTCGGTCA 203105 1 6 1CGTCACGACACCTACACAAAGCCGCACTCCCTCTGGA 203106 1 6 1GTATGGTTAACACCAATTACCGGATCCGTAGATTCA 203107 0 6 0AACAATACCACAGTGGATGCTACCAGTCCCATTAT 203108 1 6 1GACCCGTTTTGTCGTTGTATTGATATGGTACGTCTTGA 203109 1 6 1AAATCGCGACCGGGACAGTGCCGATATTTTTCACG 203110 0 6 1CTATCCGATGCTACCACTGTTTGAAATCAACAGCC 203111 1 6 0CTTCATTCGGTTCGGCCGTTCGAACACACTTGTCCCA 203112 1 6 1GCTAATGCTCGGTGAGGATCGAGCTCCGGTGCCTC 203113 1 6 1

TABLE 40 Oligonucleotide probes that bind bevacizumab respondercancer tissue Variable Region SEQ ID NO Seq name Full sequence (5′->3′)SEQ ID NO 203076 5825C-R9- /5DIG/CTAGCATGACTGCAGTACGTATATGCGATGCT 206479S1-5′DIG AGCTCGAAGCGTGTGCAGTCCCTCTGTCTCTTATACAC ATCTGACGCTGCCGACGA5825C-R9- /5DIG/TCGTCGGCAGCGTCAGATGTGTATAAGAGACA 206480 S1-RC-GAATAGGGAAATGCGACAATGATTCAAGTACCCAGTAC 5′DIG GTACTGCAGTCATGCTAG 2030775825C-R9- /5DIG/CTAGCATGACTGCAGTACGTGTCCAGCTCGCA 206481 S2-5′DIGATTACACTAGTTTGCCAGTAAAAGCTGTCTCTTATACA CATCTGACGCTGCCGACGA 5825C-R9-/5DIG/TCGTCGGCAGCGTCAGATGTGTATAAGAGACA 206482 S2-RC-GAGGGACTGCACACGCTTCGAGCTAGCATCGCATATAC 5′DIG GTACTGCAGTCATGCTAG 2030785825C-R9- /5DIG/CTAGCATGACTGCAGTACGTTCCGGAGTCCAT 206483 S3-5′DIGAAGACTACGGATAGCTTTGACCGCTGTCTCTTATACAC ATCTGACGCTGCCGACGA 5825C-R9-/5DIG/TCGTCGGCAGCGTCAGATGTGTATAAGAGACA 206484 S3-RC-GTATCTACACTTTTGTTTCCGAAACGCGATGTGCATGA 5′DIG CGTACTGCAGTCATGCTAG 2030795825C-R9- /5DIG/CTAGCATGACTGCAGTACGTCATGCACATCGC 206485 S4-5′DIGGTTTCGGAAACAAAAGTGTAGATACTGTCTCTTATACA CATCTGACGCTGCCGACGA 5825C-R9-/5DIG/TCGTCGGCAGCGTCAGATGTGTATAAGAGACA 206486 S4-RC-GAGTGTATGAGGAATCCGAGGTCACACGTCGTGTGGAC 5′DIG GTACTGCAGTCATGCTAG 2030805825C-R9- /5DIG/CTAGCATGACTGCAGTACGTCCACACGACGTG 206487 S5-5′DIGTGACCTCGGATTCCTCATACACTCTGTCTCTTATACAC ATCTGACGCTGCCGACGA 5825C-R9-/5DIG/TCGTCGGCAGCGTCAGATGTGTATAAGAGACA 206488 S5-RC-GCTTTTACTGGCAAACTAGTGTAATTGCGAGCTGGACA 5′DIG CGTACTGCAGTCATGCTAG 2030815825C-R9- /5DIG/CTAGCATGACTGCAGTACGTACTGGGTACTTG 206489 S6-5′DIGAATCATTGTCGCATTTCCCTATTCTGTCTCTTATACAC ATCTGACGCTGCCGACGA 5825C-R9-/5DIG/TCGTCGGCAGCGTCAGATGTGTATAAGAGACA 206490 S6-RC-GCGGTCAAAGCTATCCGTAGTCTTATGGACTCCGGAAC 5′DIG GTACTGCAGTCATGCTAG

Example 25 Background Optimization and Automation

In this Example, we optimized fixed tissue staining with oligonucleotideprobe libraries as in the experiments described in the Examples above.For example, such optimization was performed to reduce non-specificbackground staining with enriched oligonucleotide probe libraries whenusing polyligand histochemistry (PHC). Certain conditions were found toaddress non-specific background staining Certain changes in the stainingprotocols are shown in Table 41. The “New” and “New+” improved protocolswere optimized and automated using the Ventana Discovery platform. Inthe table, BA refers to BlockAid Blocking Solution (Thermo FisherScientific), and PBS refers to phosphate buffered saline. As seen fromthe table, the blocking conditions are more stringent in the New andNew+ protocols.

TABLE 41 Background optimization conditions Original New New+ ProtocolProtocol Protocol Permeabilization No Yes Yes (0.1% Triton-100 (0.1%Triton-100 for 10 mins) for 10 mins) Deparaffinization 69° C. for 69° C.for 69° C. for 32 mins 32 mins 32 mins Antigen Retrieval 95° C. for 95°C. for 95° C. for 32 mins 32 mins 32 mins Library cocktail 10% BA 25% BA25% BA (no Triton) (0.01% Triton) (0.01% Triton) Rinses 1 rinse 4 rinses4 rinses Str-HRP + MgCl₂ In PBS In PBS In 25% BA

The complete protocol is as follows. Examples of staining varioustissues will follow. Reagents are shown in Table 42. The equipment is aVentana Discovery Autostainer.

TABLE 42 Raw Materials Reagent Vendor cat # Salmon DNA Life Tech15632-011 tRNA Life Tech AM7119 BlockAid Life Tech B10710 StreptavidinPoly-HRP Life Tech 21140 MgCl2 Affymetrix 78641 Hyclone PBS V.W.R.International SH30256.01 Sigma PBS Packet Sigma-Aldrich P3813 H2OSigma-Aldrich W4502 Molecular Grade Water Life Tech 10977-015 DAB BufferDako SM803

Reagent Preparation

Step 1: Permeabilization of all slides immediately prior to epitoperetrieval. Completely immerse slides in 0.1% Triton X 100 in 1×PBS,incubate for 10 mins at room temperature (RT).

Step 2: Wash the slide by dipping 10× in 750 mL 1×PBS (1Liter coplingjar).

Step 3: Prepare Competitor/Block/Aptamer Library cocktail. In Table 43,the SA-poly HRP cocktail is shown. SA-poly HRP refers to StreptavidinPoly-HRP conjugate, which is biotin-binding protein conjugated withpolymers of horseradish peroxidase that enables signal amplification anddetection of biotinylated binding agents (e.g., antibodies or aptamers)for IHC and other methods.

TABLE 43 SA-poly HRP cocktail Stock Cocktail Final Concen- Concen-Volume from Concen- Reagent tration tration stock, ul tration H2O — —0.00 SA-poly HRP, x 250 4 384.0 1.00 PBS, x 20 4 4800.0 1.00 MgCl2, mM1000 12 288.0 3.00 BlockAid, % 100 77.2 18528.0 19.30

The biotinylated oligonucleotide probe library is mixed with MgCl₂ priorto addition to the Competitor/Block master mix shown in Table 44:

TABLE 44 Block/Aptamer master mix Volume of Interme- stock to Stockdiate Cocktail make Final Concen- stock Concen- cocktail, Concen-Reagent tration (in H20) tration ul tration H₂O 72.5 Triton, % 100 10.03 14.2 0.01 Salmon DNA, ng/ul — 300 1.96 3.1 0.65 tRNA, ng/μl — 3001.96 3.1 0.65 BlockAid, % 100 — 75.00 354.4 25.00 MgCl₂, mM 1000 — 9.004.3 3.00 PBS, x 20 — 0.00 0.0 0.00 Oligo library ng/μl 15 — 0.67 21.00.22

Staining

Step 4: Aptohistochemistry on Ventana Discovery autostainer

Deparaffinization and Epitope Retrieval

All Slides: Apply corresponding block/aptamer solution 150 μl (Table44), incubate for 1 h@RT (300 μl of Running buffer will be addedautomatically)

All Slides: Apply 100 μl of SA-HRP+MgCl₂+19% BA SA-poly HRP cocktail(Table 43)

Apply DAB buffer

Apply Hematoxylin

Post Staining

Step 5: Pour off liquid coverslip and use paper towel to wick offresidual oil from edge of glass.

Step 6: Standard coverslipping.

Step 7: Score slide staining.

Tissue staining

The protocol above was used to stain fixed tissues from variousanatomical locales, including tumor samples from ovarian, breast,pancreatic, bladder, melanoma, head & neck, kidney, non-small cell lung(NSCLC), glioblastoma (GBM), hepatocellular carcinoma (HCC) and colonorigin. The oligonucleotide probe library used was the R6 library fromHER2+ breast tissue enrichment in Example 19. The unenriched library(R0) was used as a control. Accordingly, the probe library was notoptimized for each tissue but nonetheless provided discernible stainingin various tissues as shown in FIGS. 19A-K. The figures show H&E stainedslides with the original protocol and the optimized protocol presentedin this Example FIG. 19A shows results for ovarian cancer samples. Thedarkest staining of all samples was observed with the original protocol,though discernible brown stain was observed in the No Library samplethat was absent any oligonucleotide probes. With the optimized library,staining was most evident with R6 and no brown staining was observed inthe No Library sample. Without being bound by theory, the stainingobserved in the absence of probe library with the original protocol maybe derived from non-specific binding of the SA-HRP, or in some cases dueto endogenous biotin. Qualitatively similar results were observed withbreast cancer tissue (FIG. 19B), pancreatic cancer tissue (FIG. 19C),bladder cancer tissue (FIG. 19D), melanoma tissue (FIG. 19E), head andneck cancer tissue (FIG. 19F), kidney cancer cancer tissue (FIG. 19G),NSCLC tissue (FIG. 19H), GBM tissue (FIG. 19I), HCC tissue (FIG. 19J),and colon cancer tissue (FIG. 19K). With melanoma and GBM tissues,slight staining was observed in the optimized no library samples, butthis staining was nonetheless noticeably reduced as compared to theoriginal no library samples. With the NCSLC and colon tissues, littlestaining was observed in the no library samples under either set ofconditions.

Example 26 Oligonucleotide Probe Enrichment on TUBB3+ Pancreatic CancerTissue

In this Example, we used the FFPE tissue enrichment protocol developedin the Examples above (see, e.g., Examples 21-22 and 25) to enrich anaïve oligonucleotide probe library against TUBB3+ pancreatic cancertissue samples.

The random ssDNA library (naïve F-TRin-35n-B 8-3s library) contains 35random nucleotides flanked by constant regions. Specifically, the naïvelibrary comprises a 5′ region (5′ CTAGCATGACTGCAGTACGT (SEQ ID NO. 4))followed by a random naïve oligonucleotide sequence of 35 nucleotidesand a 3′ region (5′ CTGTCTCTTATACACATCTGACGCTGCCGACGA (SEQ ID NO. 5)).The samples were pancreatic FFPE tissue samples with TUBB3 statusconfirmed by conventional IHC. Seven rounds of enrichment wereperformed. We initially performed enrichment with the full protocol fromExample 25 but no enrichment was observed. Without being bound bytheory, it is likely the optimized protocol in Example 25 is toostringent as it was optimized for probing and not enrichment. Thus, theoptimized protocol was adapted for the enrichment process as shown inFIG. 20A. In this scheme, enrichment was performed according to theorder P→P→P→(N→P)→(N→P)→(N→P)→P, where “P” (“Pos” in FIG. 20A) refers topositive selection against TUBB3+ samples and “N” (“Neg” in FIG. 20A)refers to negative selection against TUBB3− samples. The enrichment wasperformed with low stringency in early rounds using the conditions asshown in FIG. 20A. For example, the figure shows that the concentrationof detergent (Triton X100) and blocking agents (BlockAid) were increasedin later rounds of enrichment. Further as indicated in the figure,staining as described in Example 25 was performed after later rounds ofenrichment to observe the enrichment process. An example in shown inFIG. 20B, where much higher levels of brown stain are observed in thePositive sample as compared to the Negative sample.

The final enriched library after seven rounds of enrichment is referredto as the TUBB3− R7 library. The TUBB3−R7 library was used to probe nineTUBB3+ and nine TUBB3− pancreatic cancer tissue slides that were notused in the enrichment process (i.e., non-enrichment cases) Stainingintensity was determined by blinded pathologists. H-Scores (i.e., [1×(%cells 1+)+2×(% cells 2+)+3×(% cells 3+)]) were calculated based on thestaining intensity for both overall slide staining and nuclear stainingResults are shown in FIG. 20C, which plots total (i) or nuclear (ii)H-score for the indicated groups of samples (i.e., TUBB3+ or TUBB3−).The p-values for the differences between the groups are indicatedbeneath each plot. The H-Scores were also used to generate ROC plots andcalculate ROC AUC values, as shown in FIG. 20D. This figure providesplots for total (i) or nuclear (ii) ROC curves. The AUC value for total(i) was 0.843 and the AUC value for nuclear (ii) was 0.889. Thus, inboth cases the TUBB3−R7 library had very high performance atdifferentiating TUBB3+ and TUBB3− pancreatic cancer tissue specimens.

In Example 19, we presented an oligonucleotide probe library that wasable to distinguish HER2+ and HER2− breast cancer samples. Similarly, inthis Example, we developed an oligonucleotide probe library that wasable to distinguish TUBB3+ and TUBB3− pancreatic cancer samples.

Example 27 TTNT for Platinum/Taxane Treatment in Ovarian Cancer

In this Example, we used the FFPE tissue enrichment protocol as inExample 26 to enrich a naïve oligonucleotide probe library againstovarian cancer tissue samples that were considered as responders ornon-responders to platinum/taxane treatment. Responder(benefit)/non-responder (non-benefit) status was determined usingtime-to-next-treatment (TTNT) after platinum/taxane treatment, alsoknown as drug free interval (DFI), as described in Examples 20-22. Forthis Example, non-responders were those with DFI<6 months and responderswere those with DFI>6 months.

Methodology was similar to that in Example 26 with modificationsdescribed here. The enrichment process is outlined in FIG. 21A.Enrichment was performed according to the orderP→P→P→(2N*→P)→(2N*→P)→(2N*→P, where “P” (“Pos” in FIG. 21A) refers topositive selection against responder tissue samples, “N” (“Neg” in FIG.21A) refers to negative selection against non-responder tissue samples,and * indicates that two negative slides were used in parallel, thesupernatants were pooled and the probes were purified with Streptavidinbeads before PCR. The reverse process was also performed, whereinpositive selection was against non-responder tissue samples and negativeselection was against responder tissue samples. As in Example 26, morestringent conditions were used in later rounds of enrichment. See FIG.21A for enrichment conditions. FIG. 21B shows examples of staining withsix enriched libraries, three libraries trained toward non-respondersand three trained toward responders, as indicated. The library in FIG.21B showed the expected staining pattern with greater staining in thepositive enrichment cases (here responders). This library will be usedto probe non-enrichment ovarian cancer samples that benefit/respond ornot from platinum/taxane treatment as described above.

Example 28 Detection of Oligonucleotide Probe Binding to Tissue Samples

Protocols similar to the Examples above were used to enrich the naïveF-TRin-35n-B 8-3s library against FFPE kidney tissue slides. Thebiotinylated library after 6 rounds of enrichment was used to probefixed kidney tissue similar to Example 25. Slides were also probed withbiotinylated unenriched F-TRin-35n-B 8-3s library as a control. Theoligonucleotide probe binding was visualized as above usingStreptavidin-horse radish peroxidase (SA-HRP) (Life Technologies, cat#11207733910). Despite stringent probing conditions, notable levels ofbackground staining seen with the unenriched library control. In thisExample, we used an alternative staining protocol to visualizeoligonucleotide probe binding to the kidney samples.

Without being bound by theory, we examined whether non-specific bindingof SA-HRP to the samples could be responsible and developed an alternatevisualization methodology. Biotin-avidin/streptavidin biological assayshave many desirable characteristics such as well known methods andreagents, do not require antibodies due to the strong and specificbiotin-avidin binding, and streptavidin beads are available for pulldown experiments/immunoprecipitation. However, in some cases endogenousbiotin in the tissue could lead to problematic background binding. Inthis Example, we tested a digoxigenin (DIG) modified oligonucleotidelibrary with anti-DIG-HRP antibody detection. Unlike biotin, digoxigeninis a steroid found exclusively in the flowers and leaves of certainplants.

FIG. 22A show staining of kidney FFPE slides using the indicated DIGmodified oligonucleotide libraries and anti-DIG-HRP antibody detection.FIG. 22Ai shows a no-library control, FIG. 22Aii shows staining with 5ng of the unenriched (R0) library, FIG. 22Aiii shows staining with 5 ngof the round (R6) library, FIG. 22Aiv shows staining with 50 ng of theunenriched (R0) library, and FIG. 22Av shows staining with 50 ng of theround 6 (R6) library. All images were taken at a 20× magnification. Inthe figures, no brown staining was observed with no-library control(FIG. 22Ai) and 5 ng R0 samples (FIG. 22Aii). Only slight staining wasobserved with the 50 ng R0 samples (FIG. 22Aiv). More staining wasobserved with the 5 ng R6 samples (FIG. 22Aiii), and strong stain wasobserved when using 50 ng of the R6 library (FIG. 22Av). These dataindicate that the DIG modified oligonucleotide libraries were effectiveat eliminating background staining observed with the biotin modifiedoligonucleotide libraries in the fixed kidney tissue samples used inthis study.

We also examined whether the incubation times during enrichment wouldinfluence the enrichment process. Four different enrichments wereperformed against kidney tissue wherein the library incubation timeduring enrichment was varied at 30 min, 1 h, 2 h, and 3 h. We thenperformed anti-DIG staining with 50 ng of libraries from six rounds ofenrichment under each of the incubation conditions. We found thatincubation time correlated with staining intensity: the longer theincubation time the stronger the staining See FIG. 22B, which showsslides from six rounds of enrichment with incubation times of 30 min(FIG. 22Bi), 1 h (FIG. 22Bii), 2 h (FIG. 22Biii), and 3 h (FIG. 22Biv).

Example 29 On-Slide Oligonucleotide Probe Enrichment Against FFPE TissueLysate

In certain instances, such as in the experiments described above inExamples 19-28, paraffin blocks comprising tumor samples, or multipleslides comprising sections from such blocks are available. In suchcases, multiple slides from a single sample, including withoutlimitation multiple sections from a tumor, can be used foroligonucleotide probe library enrichment and/or probing. In thisExample, we developed a method for on-slide oligonucleotide probeenrichment against FFPE tissue lysate. Lysates from FFPE tissue slideswere arrayed onto nitrocellulose film slides for enrichment andanalysis. Such alternate methods may prove beneficial in certain cases,e.g., where limited samples are available, such as a single FFPE tissueslide per patient or tumor sample.

Methodology 2300 is outlined in FIG. 23A. As shown, 1 μL of 2.5 μg/μlFFPE lysate is arrayed onto nitrocellulose film slides (AVID FilmSlide+64-well ProPlate; Grace Bio-Labs, Bend, Oreg.) 2301. The slidesare air dried overnight (O/N) at 4° C. 2302. The slides are washed sixtimes in wash buffer comprising 50 μl of 1×PBS, 3 mM MgCl₂ 2303. Thenaïve F-Trin library as described herein is added in variousconcentrations (0.1/0.5/2.5 ng) to certain wells in 20 μl with blockingbuffer comprising 1×PBS (pH 7.4), 3 mM MgCl₂, 1% HSA, 0.5% F127, 8 ng/μlSalmon Sperm DNA and 8 ng/μl Yeast tRNA 2304. The library is incubatedon the film slides for 1 hour at room temperature (RT) with shaking at100 rpm. After incubation, the film slides are washed five times withwash buffer 2305. The wells of the film slides are scraped with a pipettip and transferred to 30 μl H₂O 2306. Oligonucleotides recovered in thescraping are amplified by asymmetric PCR as described herein and singlestranded oligonucleotide probes (ssDNA) are purified 2307. For thedesired number of rounds of enrichment, 0.1/0.5/2.5 ng of the previousround's enriched library in blocking solution is added to fresh filmslides 2308. After incubation, the oligonucleotide probes that bound tothe sample are recovered and amplified as in round 1 2309. The recoveredlibrary was sequenced using next-generation sequencing 2310 after round3.

The above procedure was performed on film slides arrayed with lysatesfrom FFPE tissue slides from human subjects with various anatomicalorigins, including breast, colon, kidney, lung and pancreas. After threerounds of enrichment, the “Rd3” libraries were sequenced as describedabove 2310. In all cases, the largest number of sequences was observedin the samples incubated with 0.1 ng of oligonucleotide library.

Three more rounds of enrichment were performed as above with breast &pancreas FFPE lysates. In one set of enrichments, the methodology 2300was as above. The library resulting from six rounds of positiveselection on lysate from breast tissue is referred to as the Br_Rd6_Lib.In a next set of enrichments, the methodology 2300 was as above withaddition of a competitor tissue lysate during incubation 2304. For thisstep in rounds 4-6, we added 0.1/0.5/2.5 ng of the 0.1 ng Rd3 libraryfrom a different tissue's enrichment in 80 μl of blocking buffer. Forexample, the Rd3 library from the breast lysate enrichments wasincubated with breast and pancreatic lysates at the same time. Thesequences bound to the breast lysate were retained, while sequencesbound to pancreatic lysate were discarded. The library resulting fromsix rounds of positive selection on lysate from breast tissue whereinthe last three rounds of enrichment included competition with lysatesfrom pancreas tissue is referred to as the Br_Rd6+_Lib.

After enrichment, we used the Br_Rd6_Lib and Br_Rd6+_Lib as well asstarting naïve F-TRin library (i.e., Rd0) to stain normal breast, colon,kidney and lung tissue slides. Slides were stained generally asdescribed in Example 40 above using a SA-HRP system using 50 ng oflibrary per slide. We observed higher level of staining when probingbreast tissue with the Br_Rd6_Lib and Br_Rd6+_Lib oligonucleotide probelibraries than with the Rd0 naïve control. Representative results areshown in FIG. 23B. Some background staining was observed in the Rd0slides (FIG. 23Bi) but much higher levels of staining with theBr_Rd6+_Lib (FIG. 23Bii) and Br_Rd6_Lib slides (FIG. 23Biii).

The above results demonstrated oligonucleotide probe library enrichmentwith selection on FFPE tissue lysate bound to film slides. We furthershowed that the enriched aptamers can stain FFPE tissue slides. See,e.g., FIG. 23B. This approach has utility if the amount of workingmaterial is limited and has the ability to perform competitive selectionusing the same library against multiple targets.

We next selected certain high abundance and high fold-changeoligonucleotide aptamers from the Rd6_Lib and Br_Rd6+_Liboligonucleotide probe libraries for use in on-slide staining of normalbreast tissue. As controls for the staining experiments, we usedoligonucleotides with complement sequences in the variable regions. Theselected sequences were synthesized with 5′-biotin. Each group ofsequences was pooled at a final concentration of 10 ng/μL. For example,eight probes with high abundance sequences selected from the Br_Rd6_Liband Br_Rd6+_Lib library were combined in a single tube and the eightcorresponding reverse complement oligonucleotides were pooled in aseparate tube. We applied 25 ng, 50 ng or 100 ng of each oligonucleotidepools to the tissue slides for staining Normal breast tissue slides fromthe same block as those used for lysate preparation were used forstaining Slides were pre-treated for 10 minutes in 0.1% Triton X-100 andrinsed in distilled H₂O. Epitope retrieval was performed at 96° C. for45 minutes. The staining protocol is as described in this Example aboveusing a streptavidin-HRP system. The selected sequences are shown inTable 45. The table indicates the variable region of the identifiedsequences. The full length sequences comprised 5′ region5′-CTAGCATGACTGCAGTACGT (SEQ ID NO 4) and a 3′ region5′-CTGTCTCTTATACACATCTGACGCTGCCGACGA (SEQ ID NO 5) surrounding thevariable region.

TABLE 45 Sequences Selected for Staining CriteriaVariable Sequence (5′->3′) SEQ ID NO High AbundanceGGGGGCCCCTTTTGTTTTCTTTTTGTTATTTTTGC 206491 (Br_Rd6_Lib andGGCTTCCTGGGGGTTTTTGTAATTGTATTTTCTGTTGA 206492 Br_Rd6+_Lib)ACCCTTTAGGTGTTTTTTTTGGTTTTCATTTTTTA 206493TTCGCCGTTTTTGTTTTGTTGTCTTAGGTTACCTC 206494 High AbundanceTGCTGGGTGGTTTGTTTTTTTATTTGGTGCATTCT 206495 (Br_Rd6_Lib)GCCGTGATTCATTTGAGGGTTCCTTGTTTGATTTTA 206496TTAGGTATGCCACGTGCCTAATTGGGGTTTTTGTTTGA 206497TGTCATCTCACCTAACCACACAACCTACTACCTCA 206498 High Fold-DifferenceTTCAATCTACACTGGTATTTCGCCTCCTCGCTGGGTGA 206499 (Br_Rd6_Lib andGGTCCTCCGGCGCATATTCCTTACCGTAAATTATA 206500 Br_Rd6+_Lib)TTGTTTCCAACTCTTGAATTTCTTGGTACTTGTCCA 206501TGGACTCTCTCCTCTGCCTCTGTGATAGCGGTTTTTGA 206502 High Fold-DifferenceCTTGAATTCCCATGTCTCTCCTGCCCCCCTCACTA 206503 (Br_Rd6_Lib)TTCTGAGGCTCACCACTTTGCACAAACTTTTCACCGA 206504GGGTTTATTCTGCTTATCCTTTCGTTTTCTTGTTGA 206505ATGCCACCACTGATCGCTAAGTTACCCCAACTGTTTGA 206506

We observed greater staining the higher the input of probes. Thus,probing with 100 ng of the oligonucleotide probes yielded the highestlevel staining, albeit with the highest levels of background stainingRepresentative results are shown in FIG. 23C and FIG. 23D (both are 20×magnifications). FIG. 23C shows results for probing with 100 ng pools ofeight sequences selected by abundance (i.e., first eight sequences inTable 45), which shows the most staining intensity. FIG. 23D showsresults for probing with 25 ng pools of eight sequences selected by highfold change (i.e., last eight sequences in Table 45), which shows lowerstaining intensity but also reduced background with the negativecontrols. In both figures, the left most panel (i) shows slides stainedwith these oligonucleotides wherein the right panel (ii) shows thestaining with their reverse complements.

Example 30 Oligonucleotide Probe Enrichment on Scraped Tissue

As another alternate enrichment scheme, in this Example we performedoligonucleotide probe library enrichment in an Eppendorf tube on tissuescraped from an FFPE slide. As in Example 29, this approach may bebeneficial in certain scenarios, including without limitation whenlimited tissue is available, such as a single FFPE slide.

We started with the naïve F-Trin library described herein. Positiveselection was performed on colon cancer tissue samples with negativeselection on non-cancer tissue from the same slide. Regions of cancerand non-cancer tissue were determined by a pathologist. Selected regionswere scraped from the slide then placed in an Eppendorf tube. Threerounds of enrichment were performed with positive selection againstcancer tissue. Five more rounds of enrichment were performed withpositive selection against cancer tissue and negative selection onnon-cancer tissue. In these experiments, separation of unbound librarywas performed by ultracentrifugation. For example, positive selectionwas performed by incubating the oligonucleotide probes with tumortissue, and collecting oligonucleotides that co-precipitated with thetissue during ultracentrifugation. Similarly, negative selection wasperformed by incubating the oligonucleotide probes with non-cancertissue, then collecting the supernatant after ultracentrifugation.

We used the library enriched after eight rounds (the “R8” library) tostain fixed tissue from four colon cancer cases used in the enrichmentprocess. We used the unenriched library (“R0”) as control Staining wasperformed with the Ventana Discovery system as in Example 25 using 50 ngof library per slide with an SA-HRP system. We compared stainingintensity on cancer and non-cancer regions from a single slide. See FIG.24A. The figure shows staining with the R0 control library on cancertissue (i), the R8 enriched library on cancer tissue (ii), the R0control library on non-cancer tissue (iii), and the R8 enriched libraryon non-cancer tissue (iv). As shown in the figure, the strongeststaining was observed on cancer tissue with enriched (round 8) libraryMinimal staining was observed on non-cancer tissue with enriched (round8) library. Finally, no staining was observed on any tissue with theunenriched (round 0) library. These results indicate the enrichmentprocess was effective to generate an oligonucleotide probe library thatcan distinguish colon cancer tissue.

We also used the R8 library to stain fixed tissue from four colon cancercases that were not used in the enrichment process. Representativeresults are shown in FIG. 24B. The figure shows staining with the R0control library on cancer tissue (i), the R8 enriched library on cancertissue (ii), the R0 control library on non-cancer tissue (iii), and theR8 enriched library on non-cancer tissue (iv). As shown in the figure,the strongest staining was observed on cancer tissue with enriched(round 8) library. For three cases we observed no staining on non-cancertissue with the enriched (round 8) library, whereas one case showedslight staining. In addition, we observed no staining on any tissue withunenriched (round 0) library, whereas the same case that showed slightstaining with the R8 library on non-cancer tissue also showed slightstaining with the R0 library as well. These results indicate that theenriched oligonucleotide probe library can distinguish colon cancertissue with high accuracy as we observed no false negatives and onepotential false positive with this sample set.

Example 31 Oligonucleotide Probe Enrichment Using Microdissection

Laser capture microdissection (LCM), also called microdissection, lasermicrodissection (LMD), or laser-assisted microdissection (LMD or LAM),is a method for isolating specific cells of interest from microscopicregions of tissue/cells/organisms. In this Example, we used LCM as amethod to extract tissue for oligonucleotide probe enrichment asdescribed herein. The method comprises dissection on a microscopic scalewith the help of a laser. LCM technology can harvest the cells ofinterest directly or can isolate specific cells by cutting away unwantedcells to give histologically pure enriched cell populations. A laser iscoupled into a microscope and focuses onto the tissue on the slide. Bymovement of the laser an element is cut out and separated from theadjacent tissue.

An extraction process follows the cutting process. In one such method, asticky surface is pressed onto the sample, which is then torn out. Thisextracts the desired region, but can also remove particles or unwantedtissue on the surface. In another method, a plastic membrane is meltedonto the sample, which is then torn out. The membrane is heated with alaser to melt it to the sample. This method may also extract undesireddebris. Extraction can also occur without contact. In one such approach,the sample is transported by laser pressure and gravity(gravity-assisted microdissection). In another approach, a cap coatedwith an adhesive is positioned directly on the thinly cut tissuesection, the section itself resting on a thin membrane (e.g.,polyethylene naphtalene). A laser gently heats the adhesive on the capfusing it to the underlying tissue and another laser cuts through tissueand underlying membrane. The membrane-tissue entity now adheres to thecap and the cells on the cap can be used in downstream applications(DNA, RNA, protein analysis).

FFPE samples are commonly fixed to glass slides. We found that not allLCM methods are appropriate for oligonucleotide probe enrichment withsuch samples. For example, LCM after epitope retrieval but beforeaddition of the oligonucleotide probe library is problematic because thesample should not be dehydrated after epitope retrieval. LCM beforeepitope retrieval can be problematic with heat induced epitope retrievalif the transfer film doesn't hold up during the standard heat epitoperetrieval. For example, we tested sample extraction using the CapSure®Macro LCM Caps and the ArcturusXT Microdissection System fromThermoFisher Scientific (Waltham, Mass.) and found that standard heatedepitope retrieval conditions (95° C.) may either melt or destroy thecapture film.

To address such issues, we performed four rounds of enrichment startingwith the naïve F-Trin library described herein on breast tissue attachedto LCM SureCaps with epitope retrieval at 75° C. We used the resultingR4 oligonucleotide probe library to stain fixed breast tissue using theDako platform as in Example 19, with epitope retrieval at 75° C. or 97°C. with between one and three wash steps after addition of library. Withepitope retrieval at 75° C. and one wash step (i.e., least stringentwash conditions), no staining was observed. Epitope retrieval at 97° C.and one wash step produced the strongest staining with the enrichedlibrary (R4). Under these conditions, we obtained slightly less stainingwith R0 library and minimal background with a no DNA control. Conditionsincluding epitope retrieval at 97° C. and stringent three wash stepsresulted in the strongest staining with enriched library (R4) andslightly less staining with the R0 library Minimal background was foundthe no DNA control but clearly less than with one wash step. We thentested both the Dako and Ventana IHC systems for staining of fixedbreast tissue. See Examples 19 and 25, respectively. We performedlibrary inputs titers (Dako: 50 ng, 25 ng and 12 ng; Ventana: 50 ng and25 ng) in combination with a stringent staining protocol, comprising 25%BlockAid in library, 19% BlockAid in SA-HRP, 0.01% Triton X-100 and fourwashes after addition of library. When using the Dako system, weobserved stronger staining with the R4 library than the unenriched R0library with all library inputs (i.e., 50 ng, 25 ng and 12 ng).Representative results are shown in FIG. 25A, which shows the samplesstained with 50 ng of the R4 library (FIG. 25Ai) or R0 control (FIG.25Aii). When using the Ventana system, we also observed strongerstaining with the R4 library than the unenriched R0 library with bothlibrary inputs (i.e., 50 ng and 25 ng). Representative results are shownin FIG. 25B, which shows the samples stained with 25 ng of the R4library (FIG. 25Bi) or R0 control (FIG. 25Bii). Under these conditions,background staining was minimal with the R0 control.

These results demonstrate enrichment of an oligonucleotide probe libraryusing microdissected FFPE tissue samples. As with Example 30, thisapproach may provide more pure samples, e.g., by dissecting purelycancer cells for analysis. As with Examples 30-31, approach may alsoprove beneficial under some conditions such as when limited sample isavailable. And as in Example 30, cancer and non-cancer samples can beextracted from a single slide.

Although preferred embodiments of the present invention have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method of enriching an oligonucleotide librarycomprising a plurality of oligonucleotides, the method comprising: (a)performing at least one round of positive selection, wherein thepositive selection comprises: (i) contacting at least one sample withthe plurality of oligonucleotides, wherein the at least one samplecomprises tissue; and (ii) recovering members of the plurality ofoligonucleotides that associated with the at least one sample; (b)performing at least one round of negative selection, wherein thenegative selection comprises: (i) contacting at least one additionalsample with the plurality of oligonucleotides, wherein at least oneadditional sample comprises tissue; (ii) recovering members of theplurality of oligonucleotides that did not associate with the at leastone additional sample; and (c) amplifying the members of the pluralityof oligonucleotides recovered in at least one or step (a)(ii) and step(b)(ii), wherein the tissue in the at least one sample in step a(i)and/or the tissue in the at least one additional sample in step b(i) islysed, or scraped from a substrate thereby enriching the oligonucleotidelibrary.
 2. The method of claim 1, wherein the recovered members of theplurality of oligonucleotides in step (a)(ii) are used as the input forthe next iteration of step (a)(i).
 3. The method of claim 1, wherein therecovered members of the plurality of oligonucleotides in step (b)(ii)are used as the input for the next iteration of step (a)(i).
 4. Themethod of claim 1, wherein the unenriched oligonucleotide librarycomprises a plurality of oligonucleotide aptamers, wherein each aptamersequence comprises a 5′ region (5′ CTAGCATGACTGCAGTACGT (SEQ ID NO. 4))followed by a random naïve aptamer sequence of 35 nucleotides and a 3′region (5′ CTGTCTCTTATACACATCTGACGCTGCCGACGA (SEQ ID NO. 5)).
 5. Themethod of claim 1, wherein the at least one sample and/or at least oneadditional sample comprise fixed tissue.
 6. The method of claim 5,wherein the fixed tissue comprises formalin fixed paraffin embedded(FFPE) tissue.
 7. The method of claim 6, wherein the FFPE tissuecomprises at least one of a fixed tissue, unstained slide, bone marrowcore or clot, biopsy sample, surgical sample, core needle biopsy,malignant fluid, and fine needle aspirate (FNA).
 8. The method of claim7, wherein the FFPE tissue is fixed on a substrate.
 9. The method ofclaim 8, wherein the at least one sample and/or the at least oneadditional sample are fixed on different substrates.
 10. The method ofclaim 8, wherein the at least one sample and/or the at least oneadditional sample is fixed on a single substrate.
 11. The method ofclaim 1, wherein the at least one sample and the at least one additionalsample differ in a phenotype of interest.
 12. The method of claim 11,wherein the at least one sample and the at least one additional sampleare from different sections of a same substrate.
 13. The method of claim12, wherein the at least one sample and the at least one additionalsample are scraped from the same substrate.
 14. The method of claim 11,wherein the phenotype comprises a tissue, anatomical origin, medicalcondition, disease, disorder, or any combination thereof.
 15. The methodof claim 14, wherein the tissue comprises muscle, epithelial, connectiveand nervous tissue, or any combination thereof.
 16. The method of claim11, wherein the phenotype comprises the presence of or likelihood ofdeveloping a tumor, neoplasm, or cancer, or characterizing the tumor,neoplasm, or cancer.
 17. The method of claim 1, further comprisingdetermining a target of the enriched members of the oligonucleotidelibrary.
 18. The method of claim 1, further comprising one or morewashing steps.
 19. The method of claim 1, further comprising epitoperetrieval prior to (a).